IL-1 receptor accessory protein

ABSTRACT

DNA encoding IL-1R AcP splice variant polypeptides, the polypeptides and methods for using the encoded polypeptides are disclosed.

[0001] This application claims the benefit under U.S.C. 119(e) of U.S.provisional application serial No. 60/244,831, filed Oct. 31, 2000. Allof which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to purified and isolated IL-1 receptorfamily members. In particular the present invention relates of DNAencoding IL-1 Receptor Accessory Protein splice variant polypeptides,polypeptides encoded by the DNA, antibodies generated against thesepolypeptides, fragmented peptides derived from these polypeptides, anduses thereof.

[0004] 2. Description of Related Art

[0005] Interleukin-1 (IL-1) is a member of a large group of cytokineswhose primary function is to mediate immune and inflammatory responses.There are several members of the IL-1 ligand family, including IL-1alpha (IL-1α), IL-1 beta (IL-1β), IL-1 eta, IL-1 receptor antagonist(IL-1ra), IL-1 delta (IL-1δ), and IL-18 (previously known as IGIF andsometimes IL-1 gamma), 1L-1 epsilon (IL-1ε), and IL-1 zeta (IL-1ζ). IL-1that is secreted by macrophages is actually a mixture of mostly IL-1βand some IL-1α (Abbas et al., 1994). IL-1β and IL-1α are the products oftwo different genes located on chromosome 2. IL-1β and IL-1α aresynthesized as precursors without leader sequences and require specificcellular proteases to process to their mature forms. Although the twoforms are less than 30 percent homologous to each other, they both bindto the same receptors and have similar activities.

[0006] IL-1ra is a biologically inactive form of IL-1 that isstructurally homologous to IL-1 and binds to the same receptors. Incontrast to IL-1β and IL-1α IL-1ra is produced with a signal sequencewhich allows for efficient secretion into the extracellular region whereit competitively competes with IL-1 for binding IL-1 receptors. (Abbaset al., 1994).

[0007] The actions of IL-1 are mediated through interaction with thetype I IL-1 receptor. IL-1 binding to the type I receptor allows theIL-1/IL-1 receptor binding complex to interact with a another protein ofsimilar structure, called IL-1 receptor accessory proteins (IL-1 AcP),to form a ternary complex. The ternary complex initiates a signalingresponse that includes the association of the cytoplasmic domains of theIL-1 receptor and IL-1R AcP with MyD88, IRAK-1, IRAK-2, IRAK-M, andTRAF6.

[0008] The biological functions of IL-1 include activating vascularendothelial cells and lymphocytes, local tissue destruction, and fever(Janeway et al., 1996). At low levels, IL-1 stimulates macrophages andvascular endothelial cells to produce IL-6, upregulates molecules on thesurface of vascular endothelial cells to increase leukocyte adhesion,and indirectly activates inflammatory leukocytes by stimulatingmononuclear phagocytes and other cells to produce certain chemokinesthat activate inflammatory leukocytes. Additionally, IL-1 is involved inother inflammatory responses such as induction of prostaglandins, nitricoxide synthetase, and metalloproteinases. These IL-1 functions arecrucial during low level microbial infections. However, if the microbialinfection escalates, IL-1 acts systemically by inducing fever,stimulating mononuclear phagocytes to produce IL-1 and IL-6, increasingthe production of serum proteins from hepatocytes, and activating thecoagulation system. IL-1 has been implicated in chronic inflammatorydiseases, such as rheumatoid arthritis and inflammatory bowel disease.There is increasing evidence that IL-1 plays a role in osteoporosis. Allof these activities are initiated by the signaling function of thecytoplasmic portion of the type I IL-1R and the IL-1RAcP. Given theimportant function of IL-1 and IL-1R, there is a need in the art foradditional cytokine receptors similar to the IL-1R family. Despite thegrowing body of knowledge, there is still a need in the art for theidentity and function of proteins involved in cellular and immuneresponses.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows results of a Taqman RNA expression analysis of avariety of tissue. The Figure illustrates that the IL-1R AcP of thisinvention is largely expressed in brain.

SUMMARY OF THE INVENTION

[0010] The present invention provides IL-1R AcP polypeptides andpolynucleotides that encode the polypeptides. The invention alsoencompasses vectors that incorporate the polynucleotides of theinvention and vectors that direct the expression of the herein describedIL-1R AcP polypeptides. Further included are host cells that incorporatethe vectors described herein and host cells that are stably ortransiently transformed or transfected with these vectors. The presentinvention further provides antibodies that specifically bind IL-1R AcPpolypeptides of this invention and fragments thereof, including thecytoplasmic domain.

[0011] In addition, the invention encompasses methods of using IL-1R AcPpolypeptides of this invention and active fragments of IL-1R AcP toscreen for antagonists and agonists of IL-1R AcP signal transduction foruse as therapeutics for diseases mediated by IL-1 family members. Theinvention also encompasses the use of sense or antisenseoligonucleotides to inhibit the expression of IL-1R AcP polypeptides.The invention further encompasses methods for the production of thesepolypeptides, including culturing a host cell under conditions promotingexpression and recovering the polypeptide from the culture medium.

[0012] Further, methods of using these polypeptides in the design ofinhibitors thereof are also an aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides human and mouse IL-1R AcPpolynucleotides and polypeptides encoded by the human and mouse IL-1RAcP polynucleotides, respectively. In one embodiment, the presentinvention provides a human IL-1R AcP polynucleotide splice variant shownin SEQ ID NO: 1, the encoded polypeptide shown in SEQ ID NO: 2, activepolypeptide fragments of the human splice variant and polynucleotidesencoding such fragments. In yet another embodiment, the presentinvention provides mouse IL-1R AcP polynucleotide splice variant shownin SEQ ID NO: 3, the encoded polypeptide shown in SEQ ID NO: 4, activepolypeptide fragments of the mouse splice variant and polynucleotidesencoding such fragments. The IL-1R AcP mouse and human nucleotidesequences and their encoded amino acid sequences are shown below: HumanIL-1R AcP polynucleotide: 1 ATGACACTTC TGTGGTGTGT AGTGAGTCTC TACTTTTATGGAATCCTGCA (SEQ ID NO:1) 51 AAGTGATGCC TCAGAACGCT GCGATGACTG GGGACTAGACACCATGAGGC 101 AAATCCAAGT GTTTGAAGAT GAGCCAGCTC GCATCAAGTG CCCACTCTTT151 GAACACTTCT TGAAATTCAA CTACAGCACA GCCCATTCAG CTGGCCTTAC 201TCTGATCTGG TATTGGACTA GGCAGGACCG GGACCTTGAG GAGCCAATTA 251 ACTTCCGCCTCCCCGAGAAC CCCATTAGTA AGGAGAAAGA TGTGCTGTGG 301 TTCCGGCCCA CTCTCCTCAATGACACTGGC AACTATACCT GCATGTTAAG 351 GAACACTACA TATTGCAGCA AAGTTGCATTTCCCTTGGAA GTTGTTCAAA 401 AAGACAGCTG TTTCAATTCC CCCATGAAAC TCCCAGTGCATAAAQTGTAT 451 ATAGAATATG GCATTCAGAG GATCACTTGT CCAAATGTAG ATGGATATTT501 TCCTTCCAGT GTCAAACCGA CTATCACTTG GTATATGGGC TGTTATAAAA 551TACAGAATTT TAATAATGTA ATACCCGAAG GTATGAACTT GAGTTTCCTC 601 ATTGCCTTAATTTCAAATAA TGGAAATTAC ACATGTGTTG TTACATATCC 651 AGAAAATGGA CGTACGTTTCATCTCACCAG GACTCTGACT GTAAAGGTAG 701 TAGGCTCTCC AAAAAATGCA GTGCCCCCTGTGATCCATTC ACCTAATGAT 751 CATGTGGTCT ATGAGAAAGA ACCAGGAGAG GAGCTACTCATTCCCTGTAC 801 GGTCTATTTT AGTTTTCTGA TGGATTCTCG CAATGAGGTT TGGTGGACCA851 TTGATGGAAA AAAACCTGAT GACATCACTA TTGATGTCAC CATTAACGAA 901AGTATAAGTC ATAGTAGAAC AGAAGATGAA ACAAGAACTC AGATTTTGAG 951 CATCAAGAAAGTTACCTCTG AGGATCTCAA GCGCAGCTAT GTCTGTCATG 1001 CTAGAAGTGC CAAAGGCGAAGTTGCCAAAG CAGCCAAGGT GAAGCAGAAA 1051 GTGCCAGCTC CAAGATACAC AGTGGAACTGGCTTGTGGTT TTGGAGCCAC 1101 AGTCCTGCTA GTGGTGATTC TCATTGTTGT TTACCATGTTTACTGGCTAG 1151 AGATGGTCCT ATTTTACCGG GCTCATTTTG GAACAGATGA AACCATTTTA1201 GATGGAAAAG AGTATGATAT TTATGTATCC TATGCAAGGA ATGCGGAAGA 1251AGAAGAATTT GTATTACTGA CCCTCCGTGG AGTTTTGGAG AATGAATTTG 1301 GATACAAGCTGTGCATCTTT GACCGAGACA GTCTGCCTGG GGGAAATACA 1351 CTCGAAGCAG TTTTTGATTTCATTCAGAGA AGCAGAAGGA TGATTGTTGT 1401 TCTGAGCCCT GACTATGTGA CAGAAAAGAGCATCAGCATG CTGGAGTTTA 1451 AACTGGQTGT CATGTGCCAG AACTCCATTG CCACCAAGCTCATTGTGGTT 1501 GAGTACCGTC CCCTTGAGCA CCCGCACCCA GGCATTCTTC AGCTCAAAGA1551 GTCTGTGTCT TTTGTGAGCT GGAAGGGAGA AAAGTCCAAA CATTCTGGCT 1601CTAAATTCTG GAAAGCTTTG CGGTTGGCTC TTCCCCTGAG AAGTCTGAGT 1651 GCCAGTTCTGGCTGGAATGA GACCTGCTCT TCCCAGTCTG ACATCAGTCT 1701 GGATCACGTT CAAAGGAGGAGAAGTCGTTT GAAAGAGCCC CCAGAACTTC 1751 AGAGCTCAGA GAGGGCTGCA GGTAGCCCTCCAGCCCCAGG CACAATGTCC 1801 AAGCACCGAG GGAACTCCTC CGCCACCTGC CGCTQTTGTGTCACCTACTG 1851 TGAAGGAQAG AATCACCTTA GGAACAAGAG CCGGGCAGAG ATTCATAACC1901 AGCCCCAGTG GGAGACACAC CTCTGTAAGC CTGTTCCCCA AGAGTCAGAA 1951ACTCAATCGA TACAAAATGG CACCACATTG GAACCCCCTG CTCCCCAGAT 2001 CTCAGCCCTTGCTCTTCATC ATTTCACGGA CTTATCCAAT AACAACGACT 2051 TTTATATCCT ATAA HumanIL-1R AcP Splice Variant encoded polypeptide: MTLLWCVVSL YFYGILQSDASERCDDWGLD TMRQIQVFED EPARIKCPLF (SEQ ID NO:2) 51 EHFLKFNYST AHSAGLTLIWYWTRQDRDLE EPINFRLPEN RISKEKDVLW 101 FRPTLLNDTG NYTCMLRNTT YCSKVAFPLEVVQKDSCFNS PMKLPVHKLY 151 IEYGIQRITC PNVDGYFPSS VKPTITWYMG CYKIQNFNNVIPEGMNLSFL 201 IALISNNGNY TCVVTYPENG RTFHLTRTLT VKVVGSPKNA VPPVIHSPND251 HVVYEKEPGE ELLIPCTVYF SFLMDSRNEV WWTIDGKKPD DITIDVTINE 301SISHSRTEDE TRTQILSIKK VTSEDLKRSY VCHARSAKGE VAKAAKVKQK 351 VPAPRYTVELACGFGATVLL VVILIVVYHV YWLEMVLFYR AHFGTDETIL 401 DGKEYDIYVS YARNAEEEEFVLLTLRGVLE NEFGYKLCIF DRDSLPGGNT 451 VEAVFDFIQR SRRMIVVLSP DYVTEKSISMLEFKLGVMCQ NSIATKLIVV 501 EYRPLEHPHP GILQLKESVS FVSWKGEKSK HSGSKFWKALRLALPLRSLS 551 ASSGWNESCS SQSDTSLDHV QRRRSRLKEP PELQSSERAA GSPPAPGTNS601 KHRGKSSATC RCCVTYCEGE NHLRNKSRAE IHNQPQWETH LCKPVPQESE 651TQWIQNGTRL EPPAPQISAL ALHHFTDLSN NNDFYIL Mouse IL-1R AcP polynucleotide1 ATGGGACTTC TGTGGTATTT GATGAGTCTG TCCTTCTATG GGATCCTGCA (SEQ ID NO:3)51 GAGTCATGCT TCGGAGCGCT GTGATGACTG GGGACTAGAT ACCATGCGAC 101 AAATCCAAGTGTTTGAAGAT GAGCCGGCTC GAATCAAGTG CCCCCTCTTT 151 GAACACTTCC TGAAGTACAACTACAGCACT GCCCATTCCT CTGGCCTTAC 201 CCTGATCTGG TACTGGACCA GGCAAGACCCGGACCTGGAG GAGCCCATTA 251 ACTTCCGCCT CCCAGAGAAT CGCATCAGTA AGGAGAAAGATGTGCTCTGG 301 TTCCGGCCCA CCCTCCTCAA TGACACGGGC AATTACACCT GCATGTTGAG351 GAACACAACT TACTGCAGCA AAGTTGCATT TCCCCTGGAA GTTGTTCAGA 401AGGACAGCTG TTTCAATTCT GCCATGAGAT TCCCAGTGCA CAAGATGTAT 451 ATTGAACATGGCATTCATAA GATCACATGT CCAAATGTAG ACGGATACTT 501 TCCTTCCAGT GTCAAACCATCGGTCACTTG GTATAAGGGT TGTACTGAAA 551 TAGTGGACTT TCATAATGTA CTACCCGAGGGCATGAACTT GAGCTTTTTC 601 ATCCCCTTGG TTTCAAATAA CGGAAATTAC ACATGTGTGGTTACATATCC 651 TGAAAACGGA CGTCTCTTTC ACCTCACCAG GACTGTGACT GTAAAGGTGG701 TGGGCTCACC AAAGGATGCA TTGCCACCCC AGATCTATTC TCCAAATGAC 751CGTGTTGTCT ATGAGAAAGA ACCAGGAGAG GAACTGGTTA TTCCCTGCAA 801 AGTCTATTTCAGTTTCATTA TGGACTCCCA CAATGAGGTC TGGTGGACCA 851 TTGATGGAAA GAAGCCTGATGACGTCACAG TCGACATCAC TATTAATGAA 901 AGTGTAAGTT ATTCTTCAAC GGAAGATGAAACAAGGACTC AGATTTTGAG 951 CATCAAGAAA GTCACCCCGG AGGATCTCAG GCGCAACTATGTCTGTCATG 1001 CTCGAAATAC CAAAGGGGAA GCTGAGCAGG CTGCCAAGGT CAAACAGAAA1051 GTCATACCAC CAAGGTACAC AGTAGAACTC GCCTGTGGTT TTGGAGCCAC 1101GGTCTTTCTG GTAGTGGTTC TCATTGTGGT TTACCATGTT TACTGGCTGG 1151 AGATGGTCCTCTTTTACCGA GCTCACTTTG GAACAGATGA AACAATTCTT 1201 GATGGAAAGC AGTATGATATTTATGTTTCC TATGCAAGAA ATGTGGAAGA 1251 AGAGGAATTT GTGCTGCTGA CGCTGCGTGGAGTTTTGGAG AATGAGTTTG 1301 GATACAAGCT GTGCATCTTC GACAGAGACA GCCTGCCTGGGGGAAATACC 1351 GTGGAAGCAG TTTTTGATTT CATTCAGAGG AGCCGAAGGA TGATTGTTGT1401 CCTGAGCCCT GACTATGTGA CAGAAAAGAG CATCAGCATG CTGGAGTTTA 1451AGCTGGGTGT CATGTGCCAG AACTCCATTG CCACTAAGCT CATTGTGGTG 1501 GAGTACCGTCCGCTTGAGCA ACCCCATCCA GGCATCATGC AGCTGAAGGA 1551 GTCTGTGTCT TTTGTAAGCTGGAAGGGAGA AAAGTCCAAA CATTCTGGCT 1601 CCAAGTTCTG GAAGGCCTTG CGTTTGGCTCTTCCCCTGAG AAGTCTGAGC 1651 GCCAGCTCCG GCTGGAATGA GAGCTGTTCT TCTCAGTCTGACATCAGTCT 1701 GGATCATGTT CAGAGGAGAA GTCGTTTGAA AGAGCCCCCA GAACTCCGAA1751 GCTCAGAGAG GGTGTCTGGA GCAGAGCCAG CCCCGGGCAC GATGTCCAAG 1801CACCGAGGGA AACCCTCAGC AGCCTGTCGC TGCTGTGTCA CCTACTGTGA 1851 AGGAGAAAGTCACCTCAGGA GCAAGAGCCG CGCAGAGATG CACACGCATC 1901 CCCAGTGGGA AACACACCTCTGTAAGCCTC CTCTCCAAGA GTCTGAAAGT 1951 CAGTGGATAC AAAATGGCAC CCGACCCGAACCCGCTCCCC AGATCTCAGC 2001 TCTTGCACTC CGCCACTTTA CAGATTTATC CAATAACAATGACTTTTATA 2051 TCCTATAA Mouse Th-1R AcP polypeptide: 1 MGLLWYLMSLSFYGILQSHA SERCDDWGLD TMRQIQVFED EPARIKCPLF (SEQ ID NO:4) 51 EHFLKYNYSTAHSSGLTLIW YWTRQDRDLE EPINFRLPEN RISKEKDVLW 101 FRPTLLNDTG NYTCMLRNTTYCSKVAFPLE VVQKDSCFNS AMRFPVHKMY 151 IEHGIHKITC PNVDGYFPSS VKPSVTWYKGCTEIVDFHNV LPEGMNLSFF 201 IPLVSNNGNY TCVVTYPENG RLFHLTRTVT VKVVGSPKDALPPQIYSPND 251 RVVYEKEPCE ELVIPCKVYF SFIMDSHNEV WWTIDGKKPD DVTVDTTINE301 SVSYSSTEDE TRTQILSIKK VTPEDLRRNY VCHARNTKGE AEQAAKVKQK 351VIPPRYTVEL ACGFGATVFL VVVLIVVYHV YWLEMVLFYR AHFGTDETIL 401 DGKEYDIYVSYARNVEEEEF VLLTLRGVLE NEFGYKLCIF DRDSLPGGNT 451 VEAVFDFIQR SRRMIVVLSPDYVTEKSISN LEFKLGVMCQ NSIATKLIVV 501 EYRPLEQPHP GIMQLKESVS FVSWKGEKSKHSGSKFWKAL RLALPLRSLS 551 ASSGWNESCS SQSDTSLDHV QRRSRLKEPP ELRSSERVSGAEPAPGTMSK 601 HRGKPSAACR CCVTYCEGES HLRSKSRAEM HTHPQWETHL CKPPLQESES651 QWIQNGTRPE PAPQISALAL RHFTDLSNNN DFYIL*

[0014] The native human IL-1R AcP of this invention includes apolymorphism that is present at about a 50/50 ratio. The polymorphismexists as an A at position 1792 of SEQ ID NO: 1, or a C at position 1792of SEQ ID NO: 1. This results in a Thr at position 598 of SEQ ID NO: 2,or a Pro at position 598 or SEQ ID NO: 2.

[0015] The human IL-1R AcP polypeptide of the present invention ishomologous to IL-1 receptor type I and its known homologs and shares anoverall 83% amino acid identity with the human IL-1R AcP described inWO96/23067, of which it is a splice variant. The IL-1R AcP polypeptidesand polynucleotides of this invention are an alternatively splicedvariant of IL-1R AcP in which part of the C-terminus of the cytoplasmicdomain is replaced by an alternative peptide sequence. At least part ofthis alternative peptide sequence is amino acids 449-687 of SEQ ID NO: 2and amino acids 449-685 of SEQ ID NO: 4, both of which are encompassedby this invention.

[0016] The IL-1R AcP polypeptides described herein are type Itransmembrane proteins with an extracellular region, a transmembraneregion and a cytoplasmic region. In one embodiment of the presentinvention, the human polypeptide of SEQ ID NO: 2 includes anextracellular region having amino acids 1-362, a transmembrane regionthat includes amino acids 363-383 and a cytoplasmic regions thatincludes amino acids 384-687. The signal peptide includes amino acids1-17 of the extracellular domain, which may alternatively include aminoacids 18-362. These regions are encoded by nucleotides as follows 1-1086encoding extracellular domain, (1-51 being the signal peptide) 1087-1149encoding the transmembrane region, and 1150-2064, encoding thecytoplasmic domain, all of SEQ ID NO: 1. The invention includespolynucleotides that encode the polypeptide of SEQ ID NO: 2 andpolynucleotides the encode the transmembrane region and the cytoplasmicregion of the human IL-1R AcP shown in SEQ ID NO: 2. Thus, for example,the present invention includes the coding region of SEQ ID NO: 1 andpolynucleotides represented by nucleotides residues 1150-2061 and exon1346-2061 of SEQ ID NO: 1

[0017] In another embodiment, the human polypeptide of SEQ ID NO: 2includes an extracellular region having amino acids 1-359, atransmembrane region that includes amino acids 360-378 and a cytoplasmicregions that includes amino acids 379-687. In this embodiment the signalpeptide includes amino acids 1-17 of the extracellular domain, which mayalternatively include amino acids 18-359. These regions are encoded bynucleotides as follows 1-1077 encoding extracellular domain, (1-51 beingthe signal peptide) 1078-1134 encoding the transmembrane region, and1135-2061, encoding the cytoplasmic domain, all of SEQ ID NO: 1. andexon 1346-2064 of SEQ ID NO: 1

[0018] Similarly, in one embodiment, the mouse IL-1R AcP splice variantof SEQ ID NO: 4 has an extracellular region that includes amino acids1-367 (1-17 being the signal peptide, so that amino acid 18-367 is alsothe extracellular region), a transmembrane region that includes aminoacids 368-388 and a cytoplasmic region that includes amino acids389-685, all of SEQ ID NO: 4. These regions are encoded by the followingnucleotide : nucleotides 1-1101, which encode the extracellular domain(1-51 encoding the signal peptide); 1102-1164 which encode thetransmembrane region; and nucleotides 1165-2058 which encode thecytoplasmic domain, all of SEQ ID NO: 3. Nucleotide residues that encodethe new exon include nucleotides 1346-2058. It is understood that thisinvention includes polynucleotides that are degenerate to the abovenoted encoding polynucleotides of SEQ ID NO: 1 and SEQ ID NO: 3Similarly, in another embodiment, the mouse IL-1R AcP splice variant ofSEQ ID NO: 4 has an extracellular region that includes amino acids 1-359(1-17 of this being the signal peptide) so that amino acid 18-359 is analternate extracellular domain, a transmembrane region that includesamino acids 360-378 and a cytoplasmic region that includes amino acids379-685, all of SEQ ID NO: 4. These regions are encoded by thefollowing: nucleotides 1-1077, which encode the extracellular domain(1-51 encoding the signal peptide); 1078-1134 which encode thetransmembrane region; and nucleotides 1135-2055 which encode thecytoplasmic domain, all of SEQ ID NO: 3. Nucleotide residues that encodethe new exon include nucleotides 1346-2055. It is understood that thisinvention includes polynucleotides that are degenerate to the abovenoted encoding polynucleotides of SEQ ID NO: 1 and SEQ ID NO: 3 Thediscovery of polynucleotides that encode the human and mouse IL-i R AcPpolypeptides described herein enables the construction of expressionvectors that incorporate polynucleotides of this invention. Thisdiscovery further enables the construction of host cells transfected ortransformed with the expression vectors, and, enables the constructionof host cells that include the encoding polynucleotides. The vectors andhost cells of the present invention are useful for expressing IL-1R AcPpolypeptides and fragments thereof that are described herein.

[0019] The actions of IL-1 family members are mediated through asignaling response that includes the association of the cytoplasmicdomains of the IL-1 receptor and/or members of the IL-1 receptor familywhich include but are not limited to SIGIRR (see WO 99/32626) TIGIRR(see WO99/32629), Xrec2 (APL) (See WO 00/36108) or IL-1R AcP with anumber of intracellular proteins which may include but are not limitedto MyD88, IRAK-1, IRAK-2, IRAK-M and TRAF6. IL-1 family members includeIL-1α, IL-1β, IL-1receptor antagonist, IL-18, and IL-1F5-IL-1F10, all asdescribed and referenced in TRENDS in immunology, Vol. 22 No. 10,October 2001. Thus, the ability to antagonize or agonize the associationof the IL-1R AcP of the present invention with one or more intracellularsignaling transduction factor proteins provides a pathway to modulatethe actions of IL-1 family members. An antagonist of a proinflammatoryIL-1 family member can be used for the inhibition and/or prevention ofdisease and medical conditions associated with that IL-1 family memberexpression. Similarly, an agonist of an IL-1 family member that has anegative or regulatory role in signaling can be used for the inhibitionand/or prevention of inflammatory disease. As described below, the IL-1RAcP polypeptides of the present invention are expressed in brain andcentral nervous system (CNS) tissue and thus play a role in modulatinginflammatory and immune responses associated with brain and CNS tissue.

[0020] In a particular embodiment, the present invention relates tocertain isolated polynucleotides that are free from contaminatingendogenous material. A “polynucleotide” refers to a molecule in the formof a separate fragment or as a component of a larger polynucleic acidconstruct. Such polynucleotides are preferably represented by and/orconstructed in the form of open reading frame uninterrupted by internalnon-translated sequences, or introns, that are typically present ineukaryotic genes. Sequences of non-translated DNA can be present 5 ′ or3 ′ from an open reading frame, where the same do not interfere withmanipulation or expression of the coding region.

[0021] Polynucleotides of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using thepolynucleotide of SEQ ID NO: 1 wherein the nucleotide at position 1792is A or C, or SEQ ID NO: 3, or a suitable fragment thereof, as a probein PCR reactions.

[0022] The polynucleotides of the invention include full-length genesand fragments that are useful as described herein. The full-length genemay include the N-terminal signal peptide or it may exclude the signalpeptide. Alternatively, full-length polynucleotides of this inventionencompass the polynucleotide excluding the native signal peptide andincorporating a different signal peptide. Suitable fragments of thepolynucleotides include those that encode the cytoplasmic domain andportions of the cytoplasmic domain that are capable of interacting with,modulating or binding signal transduction factors, including but notlimited to, MyD88, IRAK-1, IRAK-2, IRAK-M and TRAF6. Preferredpolynucleotides of the present invention are those that encode thepolypeptides of SEQ ID NO: 2, wherein the amino acid residue at position598 is a Thr or Pro, and SEQ ID NO: 4, e.g. the polynucleotides of SEQID NO: 1 and SEQ ID NO: 3, respectively, wherein the nucleotide atposition 1792 of SEQ ID NO: 1 is A or C. Useful polynucleotide fragmentsof the present invention include those that encode a cytoplasmic domainof SEQ ID NO: 2 and SEQ ID NO: 4, i.e. amino acid residues 384-687 ofSEQ ID NO: 2, wherein the amino acid residue at position 598 is Thy orPro, and amino acid residues 389-685 of SEQ ID NO: 4. Embodiments ofnucleotides that encode the cytoplasmic domain of the describedpolypeptides include nucleotide residues 1150-2064 of SEQ ID NO: 1,wherein the nucleotide at position 1792 is A or C, and 1165-2058 of SEQID NO: 3.

[0023] Additional useful polynucleotide fragments of the presentinvention include those that encode amino acid residues 379-687 of SEQID NO: 2, wherein the amino acid residue at position 598 is Thy or Pro,and amino acid residues 379-685 of SEQ ID NO: 4. Another fragmentincludes amino acids 649-687 of SEQ ID NO: 2 and amino acids 649-685 ofSEQ ID NO: 4. Embodiments of nucleotides that encode just describedpolypeptides include nucleotide residues 1135-2061 of SEQ ID NO: 1 and1346-2061 of SEQ ID NO: 1, wherein the nucleotide at position 1792 is Aor C, and 1165-2058 and nucleotide 1346-2055, both of SEQ ID NO: 3.

[0024] Polynucleotides of this invention include those that aredegenerate to SEQ ID NO: 1, SEQ ID NO: 3 and fragments that encodebiologically active polypeptide, including those described above.Degenerate polynucleotides arise because of the known degeneracy of thegenetic code, wherein more than one codon can encode the same aminoacid, a polynucleotide can vary from that shown in the coding sequencesof SEQ ID NO: 1 and SEQ ID NO: 3 and still encode a polypeptide havingthe amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.Such variant polynucleotides can result from silent mutations (e.g.,occurring during PCR amplification), or can be the product of deliberatemutagenesis of a native sequence.

[0025] The present invention thus provides isolated DNAs that include(a) SEQ ID NO: 1 wherein the nucleotide at position 1792 is A or C; (b)SEQ ID NO: 3; (c) DNA encoding the polypeptide of SEQ ID NO: 2, whereinthe amino acid residue at 598 is Pro or Thr.; (d) DNA encoding thepolypeptide of SEQ ID NO: 4; (e) DNA encoding the polypeptide havingamino acid residues 449-687 of SEQ ID NO: 2, wherein the residue at 598is Thr or Pro; (f) ) DNA encoding a polypeptide having amino acidresidues 449-685 of SEQ ID NO: 4, (g) DNA encoding a polypeptide havingamino acid residues 384-687 of SEQ ID NO: 2, wherein the amino acidresidue at 598 is Pro or Thr; (h) DNA encoding amino acid residues379-687 of SEQ ID NO: 2, wherein the amino acid residues at 598 is Proor Thr, (i) DNA encoding a polypeptide having amino acid residues389-685 of SEQ ID NO: 4; (j) DNA encoding a polypeptide having aminoacid residues 379-685 of SEQ ID NO: 4 (k) DNA encoding a polypeptidethat is a fragment of the polypeptide having amino acid residues 384-687of SEQ ID NO: 2, wherein the amino acid residue at 598 is Thr or Pro;the fragment capable of interacting with an intracellular signaltransduction factor; (l) DNA encoding a polypeptide that is a fragmentof the polypeptide having amino acids residues 379-687 of SEQ ID NO: 2,wherein the amino acid residue at 598 is Thr or Pro, the fragmentcapable of interacting with an intracellular signal transduction factor;(m) DNA encoding a polypeptide that is a fragment of the polypeptidehaving amino acid residues 389-685 of SEQ ID NO: 4; the fragment capableof interacting with an intracellular signal transduction factor; (n) DNAencoding a polypeptide that is a fragment of the polypeptide havingamino acid residues 379-685 of SEQ ID NO: 4, the fragment capable ofinteracting with an intracellular signal transduction factor; (o) DNAthat is the complement of a DNA capable of hybridization to a DNA of (a)through (n) under conditions of moderate stringency; and, (p) DNA whichis degenerate as a result of the genetic code to a DNA defined in(a)-(o).

[0026] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C., and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0027] The present invention further includes DNA encoding polypeptidefragments and polypeptides comprising inactivated N-glycosylationsite(s), inactivated protease processing site(s), or conservative aminoacid substitution(s), as described below.

[0028] In another embodiment, the polynucleotides of this inventioninclude variant polynucleotides that are at least 85% identical to thepolynucleotides that encode the cytoplasmic domain of SEQ ID NO: 2 andSEQ ID NO: 4. Similarly, the present invention includes polynucleotidesthat are at least 80% identical to polynucleotides that encodepolypeptide fragments of the cytoplasmic domain of SEQ ID NO: 2 and SEQID NO: 4 when the polypeptide fragments are capable of interacting witha signal transduction factor, including but not limited to MyD88,IRAK-1, IRAK-2, IRAK-M, and TRAF6.

[0029] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

[0030] The polynucleotides of this invention are useful in processes forpreparing polypeptides that are disclosed herein. For example, a DNAencoding a polypeptide of the present invention, or polypeptide fragmentof this invention may be subcloned into an expression vector forproduction of the polypeptide or polypeptide fragment. The DNAadvantageously is fused to a polynucleotide encoding a suitable leaderor signal peptide. Alternatively, a polynucleotide described herein orsuitable fragment may be chemically synthesized using known techniques.DNA fragments may be prepared by restriction endonuclease digestion of afull length cloned DNA sequence, and isolated by electrophoresis onagarose gels. If necessary, oligonucleotides that reconstruct the 5 ′ or3 ′ terminus to a desired point may be ligated to a DNA fragmentgenerated by restriction enzyme digestion. Such oligonucleotides mayadditionally contain a restriction endonuclease cleavage site upstreamof the desired coding sequence, and position an initiation codon (ATG)at the N-terminus of the coding sequence.

[0031] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA encoding a desired polypeptidefragment. Oligonucleotides that define the desired termini of the DNAfragment are employed as 5 ′ and 3 ′ primers. The oligonucleotides maycontain recognition sites for restriction endonucleases, to facilitateinsertion of the amplified DNA fragment into an expression vector. PCRtechniques are described in Saiki et al., Science 239:487 (1988);Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., SanDiego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press, Inc. (1990).

[0032] IL-1R AcP polypeptides of the present invention include thosethat are naturally occurring and variant polypeptides that are producedthrough various techniques such as procedures involving recombinant DNAtechnology. For example, DNAs encoding polypeptides described herein canbe derived from SEQ ID NO: 1 and/or SEQ ID NO: 3 as described above, byin vitro mutagenesis, which includes site-directed mutagenesis, randommutagenesis, and in vitro nucleic acid synthesis. Such forms include,but are not limited to, derivatives, variants, and oligomers, as well aspolynucleotides that encode fusion proteins or fragments thereof.

[0033] The polypeptides of the invention include full-length proteinsencoded by the polynucleotides set forth above. A preferred humanpolypeptide includes the amino acid residues of SEQ ID NO: 2, whereinthe amino acid residue at 598 is Pro or Thr; and, a preferred mousepolypeptide includes the amino acid residues of SEQ ID NO: 4. Further,the invention encompasses fragments and domains of the polypeptides ofSEQ ID NO: 2 and SEQ ID NO: 4 including the cytoplasmic domains of SEQID NO: 2 and SEQ ID NO: 4. In particular such fragments include aminoacids 384-687 of SEQ ID NO: 2, amino acids 379-687 of SEQ ID NO: 2,wherein the amino acids residue at 598 is Pro or Thr; amino acids389-685 of SEQ ID NO: 4, amino acids 379-685 of SEQ ID NO: 4;polypeptide fragments of amino acids 384-687 of SEQ ID NO: 2,polypeptide fragments of amino acid residues 379-687 of SEQ ID NO: 2;polypeptide fragments of amino acids 389-685 of SEQ ID NO: 4,polypeptide fragments of amino acids residues 379-685 of SEQ ID NO: 4,where the above mentioned fragments interact with a intracellular signaltransduction factor. Other useful fragments include amino acids 449-687of SEQ ID NO: 2 and amino acids 449-685 of SEQ ID NO: 4.

[0034] The polypeptides of the invention may be membrane bound or theymay be secreted and thus soluble. Soluble polypeptides are capable ofbeing secreted from the cells in which they are expressed. In general,soluble polypeptides may be identified (and distinguished fromnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

[0035] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the polypeptide449-687 of SEQ ID NO: 2 and the polypeptide of 449-685 of SEQ ID NO: 4.The position at 598 of SEQ ID NO: 2 may be Pro or Thr. As discussedbelow and mentioned above, fragments derived from the cytoplasmic domainfind use in studies of signal transduction, and in regulating cellularprocesses associated with transduction of biological signals.Polypeptide fragments also may be employed as immunogens, in generatingantibodies.

[0036] A polypeptide variant as referred to herein means a polypeptidesubstantially homologous to native IL-1R AcP polypeptide describedherein, but which has an amino acid sequence different from that of thedisclosed native IL-1R AcP polypeptides because of one or moredeletions, insertions, or substitutions. A variant polypeptide has anamino acid sequence that preferably is at least 85% identical to anative polypeptide amino acid sequence, most preferably at least 90%identical. The percent identity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

[0037] Variants also include embodiments in which a polypeptide orfragment comprises an amino acid sequence that is at least 85%identical, at least 90% identical, at least 95% identical, and at least98% identical to preferred polypeptide or fragment thereof. Such variantfurther retains an activity that is characteristic of the IL-1R AcP ofthis invention. One example is a variant that interacts with an IL-1receptor family member or IL-1 in a signaling complex. Another exampleis a polypeptide or this invention, including fragments and variantshaving the capacity to interact with or modulate intracellular signaltransduction factors, including IRAK-1, IRAK-2, IRAK-M, and TRAFs. Yet,another example is a variant that retains similar binding affinity orsimilar as an IL-1R AcP polypeptide of this invention, includingfragments and variants. Binding affinity can be measured by conventionalprocedures, e.g., as described in U.S. Pat. No. 5,512,457. Similarly,variant cytoplasmic domain polypeptides include polypeptides that retainat least an 80% amino acid identity with the cytoplasmic domain of SEQID NO: 2 or SEQ ID NO: 4 and which interact with signal transductionfactors.

[0038] Percent identity may be determined as above. Alternatively, thepercent identity of two protein sequences can be determined by comparingsequence information using the GAP computer program, based on thealgorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a scoring matrix, blosum62, as described by Henikoff and Henikoff(Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3)a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by one skilled in the art of sequence comparison may alsobe used.

[0039] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0040] As stated above, the invention provides isolated and purified, orhomogeneous, IL-1R AcP polypeptides, both recombinant andnon-recombinant. Variants and derivatives of native polypeptides thatretain the desired biological activity can be obtained by mutations ofnucleotide sequences coding for native IL-1R AcP polypeptides of thisinvention. Alterations of the native amino acid sequence can beaccomplished by any of a number of conventional methods. Mutations canbe introduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

[0041] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene, whereinpredetermined codons can be altered by substitution, deletion, orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,all of which are incorporated by reference.

[0042] Polypeptides can be modified to create polypeptide derivativesencompassed by this invention by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups andthe like. Covalent derivatives of the disclosed IL-1R AcP polypeptidescan be prepared by linking the chemical moieties to functional groups onIL-1R AcP amino acid side chains or at the N-terminus or C-terminus of apolypeptide or the extracellular domain thereof. Other derivatives ofthe native polypeptides described herein within the scope of thisinvention include covalent or aggregative conjugates of nativepolypeptides or their fragments with other proteins or polypeptides,such as by synthesis in recombinant culture as N-terminal or C-terminalfusions. For example, the conjugate can comprise a signal or leaderpolypeptide sequence (e.g. the α-factor leader of Saccharomyces) at theN-terminus of an IL-1R AcP polypeptide. The signal or leader peptideco-translationally or post-translationally directs transfer of theconjugate from its site of synthesis to a site inside or outside of thecell membrane or cell wall.

[0043] Conjugates comprising diagnostic (detectable) or therapeuticagents attached thereto are contemplated herein, as discussed in moredetail below.

[0044] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys,which is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the American Type CultureCollection under accession no. HB 9259. Monoclonal antibodies that bindthe FLAG® peptide are available from Eastman Kodak Co., ScientificImaging Systems Division, New Haven, Conn.

[0045] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence. A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0046] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0047] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0048] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues, or deletions ofterminal or internal residues are encompassed by the invention. Forexample, N-glycosylation sites in the polypeptide extracellular domaincan be modified to preclude glycosylation, allowing expression of areduced carbohydrate analog in mammalian and yeast expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Yis Ser or Thr. Appropriate substitutions, additions, or deletions to thenucleotide sequence encoding these triplets will result in prevention ofattachment of carbohydrate residues at the Asn side chain. Alteration ofa single nucleotide, chosen so that Asn is replaced by a different aminoacid, for example, is sufficient to inactivate an N-glycosylation site.Alternatively, the Ser or Thr can by replaced with another amino acid,such as Ala. Known procedures for inactivating N-glycosylation sites inproteins include those described in U.S. Pat. No. 5,071,972 and EP276,846, hereby incorporated by reference.

[0049] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0050] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0051] Encompassed by the invention are oligomers or fusion proteinsthat contain the polypeptides, variants and fragments, described herein.Such oligomers may be in the form of covalently-linked ornon-covalently-linked multimers, including dimers, trimers, or higheroligomers. In one aspect of the invention, the oligomers maintainsimilar ternary complex binding ability of the polypeptide componentsand provide therefor, bivalent, trivalent, etc., binding sites.

[0052] One embodiment of the invention is directed to oligomerscomprising multiple polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of the polypeptides attached thereto, asdescribed in more detail below.

[0053] As one alternative, an oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn etal. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992).

[0054] One fusion protein embodiment of the present invention isdirected to a dimer comprising two fusion proteins created by fusing apolypeptide of the invention to an Fc polypeptide derived from anantibody. A gene fusion encoding the polypeptide/Fc fusion protein isinserted into an appropriate expression vector. Polypeptide/Fc fusionproteins are expressed in host cells transformed with the recombinantexpression vector, and allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between the Fc moieties toyield divalent molecules.

[0055] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fc region of an antibodycomprising any or all of the CH domains of the Fc region. Truncatedforms of such polypeptides containing the hinge region that promotesdimerization are also included. Preferred polypeptides comprise an Fcpolypeptide derived from a human IgG1 antibody.

[0056] One suitable Fc polypeptide, described in PCT application WO93/10151 (hereby incorporated by reference), is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and inBaum et al., (EMBO J. 13:3992-4001, 1994) incorporated herein byreference. The amino acid sequence of this mutein is identical to thatof the native Fc sequence presented in WO 93/10151, except that aminoacid 19 has been changed from Leu to Ala, amino acid 20 has been changedfrom Leu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fc receptors.

[0057] The above-described fusion proteins comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

[0058] In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourpolypeptide extracellular regions.

[0059] Alternatively, the oligomer is a fusion protein comprisingmultiple polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA encoding a desired peptide linker may be insertedbetween, and in the same reading frame as, the DNA encoding apolypeptide of the invention, using any suitable conventional technique.For example, a chemically synthesized oligonucleotide encoding thelinker may be ligated between the sequences. In particular embodiments,a fusion protein comprises from two to four polypeptides of thisinvention, separated by peptide linkers.

[0060] Another method for preparing the oligomers of the inventioninvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, 1988), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize.

[0061] The zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The products of the nuclear oncogenes fos andjun comprise zipper domains that preferentially form heterodimer (O'Sheaet al., Science 245:646, 1989, Turner and Tjian, Science 243:1689,1989). The zipper domain is necessary for biological activity (DNAbinding) in these proteins.

[0062] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton,Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and HumanRetroviruses 6:703, 1990). The zipper domains in these fusogenic viralproteins are near the transmembrane region of the proteins; it has beensuggested that the zipper domains could contribute to the oligomericstructure of the fusogenic proteins. Oligomerization of fusogenic viralproteins is involved in fusion pore formation (Spruce et al, Proc. Natl.Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been recentlyreported to play a role in oligomerization of heat-shock transcriptionfactors (Rabindran et al., Science 259:230, 1993).

[0063] Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

[0064] Certain leucine zipper moieties preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, 1994) and inU.S. Pat. No. 5,716,805, hereby incorporated by reference in theirentirety. This lung SPD-derived leucine zipper peptide comprises theamino acid sequence Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala LeuGln Gly Gln Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr.

[0065] Another example of a leucine zipper that promotes trimerizationis a peptide comprising the amino acid sequence Arg Met Lys Gln Ile GluAsp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu Arg, as described in U.S. Pat. No.5,716,805. In one alternative embodiment, an N-terminal Asp residue isadded; in another, the peptide lacks the N-terminal Arg residue.

[0066] Fragments of the foregoing zipper peptides that retain theproperty of promoting oligomerization may be employed as well. Examplesof such fragments include, but are not limited to, peptides lacking oneor two of the N-terminal or C-terminal residues presented in theforegoing amino acid sequences. Leucine zippers may be derived fromnaturally occurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

[0067] Other peptides derived from naturally occurring trimeric proteinsmay be employed in preparing trimeric IL-1R AcP of this invention.Alternatively, synthetic peptides that promote oligomerization may beemployed. In particular embodiments, leucine residues in a leucinezipper moiety are replaced by isoleucine residues. Such peptidescomprising isoleucine may be referred to as isoleucine zippers, but areencompassed by the term “leucine zippers” as employed herein.

[0068] Art recognized methods for expressing, isolating and purifyingpolypeptides are suitable for preparing and purifying polypeptides andpolypeptide fragments of the present invention. In general, methods forproducing polypeptides include the steps of culturing host cells thatincorporate or a transfected with a an expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0069] Vectors and host cells that incorporate polynucleotides of thepresent invention are within the scope of the present invention. DNAencoding a polypeptide or fragment of the invention, that isincorporated in vectors is operably linked to suitable transcriptionalor translational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are operably linked when the regulatory sequencefunctionally relates to the DNA sequence. Thus, a promoter nucleotidesequence is operably linked to a DNA sequence if the promoter nucleotidesequence controls the transcription of the DNA sequence. An origin ofreplication that confers the ability to replicate in the desired hostcells, and a selection gene by which transformants are identified, aregenerally incorporated into the expression vector.

[0070] In addition, a polynucleotide encoding an appropriate signalpeptide (native or heterologous) can be incorporated into expressionvectors. A DNA sequence for a signal peptide (secretory leader) may befused in frame to the nucleic acid sequence of the invention so that theDNA is initially transcribed, and the mRNA translated, into a fusionprotein comprising the signal peptide. A signal peptide that isfunctional in the intended host cells promotes extracellular secretionof the polypeptide. The signal peptide is cleaved from the polypeptideupon secretion of polypeptide from the cell.

[0071] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site. Thus, particular embodiments of mature proteinsprovided herein include, but are not limited to, proteins having theN-terminal residue at the predicted cleavage of the signal peptide,amino acid 17-18 of SEQ ID NO: 2 and amino acid 17-18 of SEQ ID NO: 4 asthe N-terminal or C-terminal amino acid.

[0072] Suitable host cells for expressing polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0073] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.In a prokaryotic host cell, such as E. coli, a polypeptide may includean N-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

[0074] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0075] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0076] IL-1R AcP encoding DNA of the present invention may be clonedin-frame into the multiple cloning site of an ordinary bacterialexpression vector. Ideally the vector would contain an induciblepromoter upstream of the cloning site, such that addition of an inducerleads to high-level production of the recombinant protein at a time ofthe investigator's choosing. For some proteins, expression levels may beboosted by incorporation of codons encoding a fusion partner (such ashexahistidine) between the promoter and the gene of interest. Theresulting “expression plasmid” may be propagated in a variety of strainsof E. coli.

[0077] For expression of the recombinant protein, the bacterial cellsare propagated in growth medium until reaching a pre-determined opticaldensity. Expression of the recombinant protein is then induced, e.g. byaddition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activatesexpression of proteins from plasmids containing a lac operator/promoter.After induction (typically for 1-4 hours), the cells are harvested bypelleting in a centrifuge, e.g. at 5,000×G for 20 minutes at 4° C.

[0078] For recovery of the expressed protein, the pelleted cells may beresuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and thenpassed two or three times through a French press. Most highly expressedrecombinant proteins form insoluble aggregates known as inclusionbodies. Inclusion bodies can be purified away from the soluble proteinsby pelleting in a centrifuge at 5,000×G for 20 minutes, 4° C. Theinclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% TritonX-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTTf.Any material that cannot be dissolved is removed by centrifugation(10,000×G for 20 minutes, 20° C.). The protein of interest will, in mostcases, be the most abundant protein in the resulting clarifiedsupernatant. This protein may be “refolded” into the active conformationby dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl₂/5 mM Zn(OAc)₂/1 mMGSSG/0. 1 mM GSH. After refolding, purification can be carried out by avariety of chromatographic methods, such as ion exchange or gelfiltration. In some protocols, initial purification may be carried outbefore refolding. As an example, hexahistidine-tagged fusion proteinsmay be partially purified on immobilized Nickel.

[0079] While the preceding purification and refolding procedure assumesthat the protein is best recovered from inclusion bodies, those skilledin the art of protein purification will appreciate that many recombinantproteins are best purified out of the soluble fraction of cell lysates.In these cases, refolding is often not required, and purification bystandard chromatographic methods can be carried out directly.

[0080] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2□ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0081] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3 ′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0082] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0083] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0084] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0085] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture,1990, pp. 15-69). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:42164220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0086] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0087] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534and PCT Application WO 97/25420) and the tripartite leader (TPL) and VAgene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491, 1982). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow, Current Opinion in Genetics and Development3:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2697-2700,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150-161, 1997, and p2A5Idescribed by Morris et al., Animal Cell Technology, 1997, pp. 529-534.

[0088] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

[0089] Additional useful expression vectors, pFLAG® and pDC311, can alsobe used. FLAG® technology is centered on the fusion of a low molecularweight (1 kD), hydrophilic, FLAG® marker peptide to the N-terminus of arecombinant protein expressed by pFLAG® expression vectors. pDC311 isanother specialized vector used for expressing proteins in CHO cells.pDC311 is characterized by a bicistronic sequence containing the gene ofinterest and a dihydrofolate reductase (DHFR) gene with an internalribosome binding site for DHFR translation, an expression augmentingsequence element (EASE), the human CMV promoter, a tripartite leadersequence, and a polyadenylation site.

[0090] Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (L-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-I receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

[0091] The invention also includes methods of isolating and purifyingthe polypeptides and fragments thereof. An isolated and purifiedpolypeptide according to the invention can be produced by recombinantexpression systems as described above or purified from naturallyoccurring cells. Polypeptides can be substantially purified, asindicated by a single protein band upon analysis by SDS-polyacrylamidegel electrophoresis (SDS-PAGE).

[0092] The expression “isolated and purified” as used herein means thata IL-1R AcP polypeptide of this invention is essentially free ofassociation with other, proteins, or polypeptides, for example, as apurification product of recombinant host cell culture or as a purifiedproduct from a non-recombinant source. The term “substantially purified”as used herein refers to a mixture that contains a polypeptide and isessentially free of association with other DNA, proteins, orpolypeptides, but for the presence of known DNA or proteins that can beremoved using a specific antibody, and which substantially purifiedproteins retain biological activity. The term “purified polypeptide”refers to either the “isolated and purified” form of the polypeptide orthe “substantially purified” form of the polypeptide, as both aredescribed herein.

[0093] The term “biologically active” as it refers to a IL-1R AcPpolypeptide or a IL-1R AcP polypeptide fragment of this invention, meansthat the polypeptide, or polypeptide fragment, is capable of associatingwith an IL-1 receptor or an IL-1 receptor family member, or that theIL-1R AcP polypeptide or fragment interacts with intracellular signaltransduction proteins.

[0094] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

[0095] With respect to any type of host cell, procedures for purifying arecombinant polypeptide or fragment will vary according to such factorsas the type of host cells employed and whether or not the recombinantpolypeptide or fragment is secreted into the culture medium. In general,the recombinant polypeptide or polypeptide fragment can be isolated fromthe host cells if not secreted, or from the medium or supernatant ifsoluble and secreted, followed by one or more concentration,salting-out, ion exchange, hydrophobic interaction, affinitypurification or size exclusion chromatography steps. As to specific waysto accomplish these steps, the culture medium first can be concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The anion exchange matrices may beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. Alternatively, a cation exchange resin can beused for purification, in which case suitable cation exchangers areinsoluble matrices having sulfopropyl or carboxymethyl functionalgroups. Other purification techniques useful in the processes of thisinvention include chromatofocusing procedures or hydrophobic interactionchromatography or affinity chromatography with a matrix whichselectively binds the recombinant protein. Examples of such resinsemployed are lectin columns, dye columns, and metal-chelating columns.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel or polymer resin having pendant methyl, octyl,octyldecyl or other aliphatic groups) can be employed to further purifythe polypeptides. Some or all of the foregoing purification steps, invarious combinations, are well known and can be employed to provide anisolated and purified recombinant protein.

[0096] Recombinant protein produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0097] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

[0098] In this aspect of the invention, polypeptide-binding proteins,such as the anti-polypeptide antibodies of the invention or otherproteins that may interact with the polypeptide of the invention, can bebound to a solid phase support such as a column chromatography matrix ora similar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of polypeptide-binding proteins of the invention to a solidphase contacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0099] Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

[0100] In the methods described above, suitable IL-1R AcP bindingpolypeptides are anti-IL-1R AcP antibodies and other proteins that arecapable of high-affinity binding of the expressed polypeptide.

[0101] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0102] The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind a IL-1R and/or a IL-1R family member orits ability to form a ternary complex with IL-1R and IL-1 in anysuitable assay, such as a conventional binding assay. To illustrate, thesample polypeptide may be labeled with a detectable reagent (e.g., aradionuclide, chromophore, enzyme that catalyzes a colorimetric orfluorometric reaction, and the like). The labeled polypeptide iscontacted with cells expressing an IL-1R and/or IL-1. The cells then arewashed to remove unbound labeled polypeptide, and the presence ofcell-bound label is determined by a suitable technique, chosen accordingto the nature of the label.

[0103] One example of a binding assay procedure is as follows. Arecombinant expression vector containing IL-1R cDNA is constructed, forexample, fusing the extracellular domain of an IL-1R to the IgG-I Fc(mutein form) as previously described for OX40/Fc (Baum et al., EMBO J.13:39924001, 1994). CV1-EBNA-1 cells in 10 cm² dishes are transfectedwith the recombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL10478) constitutively express EBV nuclear antigen-1 driven from the CMVimmediate-early enhancer/promoter. CV1-EBNA-1 was derived from theAfrican Green Monkey kidney cell line CV-1 (ATCC CCL 70), as describedby McMahan et al. (EMBO J. 10:2821, 1991).

[0104] The transfected cells are cultured for 24 hours, and the cells ineach dish then are split into a 24-well plate. After culturing anadditional 48 hours, the transfected cells (about 4×10⁴ cells/well) arewashed with BM-NFDM, which is binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) towhich 50 mg/ml nonfat dry milk has been added. The cells then areincubated for 1 hour at 37° C. with various concentrations of, forexample, a soluble polypeptide/Fc fusion protein made as set forthabove. Cells then are washed and incubated with a constant saturatingconcentration of a ¹²⁵I-mouse anti-human IgG in binding medium, withgentle agitation for 1 hour at 37° C. After extensive washing, cells arereleased via trypsinization.

[0105] The mouse anti-human IgG employed above is directed against theFc region of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵I-antibody is assayed in theabsence of the Fc fusion protein, as well as in the presence of the Fcfusion protein and a 200-fold molar excess of unlabeled mouse anti-humanIgG antibody.

[0106] Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogammacounter. Affinity calculations (Scatchard, Ann. N. Y. Acad. Sci. 51:660,1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer.

[0107] Another type of suitable binding assay is a competitive bindingassay. To illustrate, biological activity of a variant may be determinedby assaying for the variant's ability to compete with the native proteinfor binding to an IL-1 receptor or IL-1 receptor family member.

[0108] Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude a radiolabeled IL-1R, IL-1 and intact cells expressing IL-1RAcP(endogenous or recombinant) on the cell surface. For example, aradiolabeled soluble IL-1R AcP fragment of the present invention can beused to compete with a soluble IL-1R AcP variant for binding to cellsurface (binding partner). Instead of intact cells, one could substitutea soluble IL-1R/Fc fusion protein bound to a solid phase through theinteraction of Protein A or Protein G (on the solid phase) with the Fcmoiety. Chromatography columns that contain Protein A and Protein Ginclude those available from Pharmacia Biotech, Inc., Piscataway, N.J.

[0109] Another type of competitive binding assay utilizes radiolabeledIL-1R such as a soluble an Fc fusion protein, and intact cellsexpressing IL-1R AcP of this invention. Qualitative results can beobtained by competitive autoradiographic plate binding assays, whileScatchard plots (Scatchard, Ann. N. Y. Acad. Sci. 51:660, 1949) may beutilized to generate quantitative results.

[0110] In addition to being used to express polypeptides as describedabove, the nucleic acids of the invention, including DNA, andoligonucleotides thereof can be used:

[0111] as probes to identify polynucleotides encoding proteins havingIL-1R

[0112] AcP activity;

[0113] as single-stranded sense or antisense oligonucleotides, toinhibit expression of polypeptide encoded by the IL-1R AcPpolynucleotides of this invention;

[0114] Fragment that are useful as probes or primers generally includeat least about 17 contiguous nucleotides of a polynucleotide thatencodes a fragment of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.In other embodiments, a fragment comprises at least 30, or at least 60,contiguous nucleotides of an similarly encoding polypeptide.

[0115] Because homologs of SEQ ID NO: 1 from other mammalian species arecontemplated herein, probes based on the DNA sequence of SEQ ID NO: 1may be used to screen cDNA libraries derived from other mammalianspecies, using conventional cross-species hybridization techniques.

[0116] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0117] Other useful fragments of the polynucleotides of this inventioninclude antisense or sense oligonucleotides comprising a single-strandednucleic acid sequence (either RNA or DNA) capable of binding to targetmRNA (sense) or DNA (antisense). Antisense or sense oligonucleotides,according to the present invention, include polynucleotide fragments ofSEQ ID NO: 1 and/or SEQ ID NO: 3. Such fragments generally comprises atleast about 14 nucleotides, preferably from about 14 to about 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0118] Binding of antisense or sense oligonucleotides to target nucleicacids results in the formation of duplexes that block or inhibit proteinexpression by one of several means, including enhanced degradation ofthe mRNA by RNAseH, inhibition of splicing, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

[0119] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0120] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, lipofection, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus.

[0121] Sense or antisense oligonucleotides are preferably introducedinto a cell containing the target polynucleotides by insertion of thesense or antisense oligonucleotide into a suitable retroviral vector,then contacting the cell with the retrovirus vector containing theinserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

[0122] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide by formation of a conjugate with aligand binding molecule, as described in WO 91/04753. Suitable ligandbinding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

[0123] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase to amonoclonal antibody targeted to a specific cell type.

[0124] Polypeptides of the present invention have a variety of usesincluding, but not limited to: therapeutic agents for diseases mediatedby IL-1 family members, models for rational drug design, targets inscreening assays directed to the discovery of inhibitors or enhancers ofIL-1R AcP signaling pathways, and antigens in antibody preparation. Asdemonstrated in Example 1, IL-1R AcP polypeptides of this invention areexpressed in the brain and central nervous system tissue. Accordingly,II-1R AcP polypeptides, IL-1R AcP polypeptides fragments, agonists andantagonists of IL-1R AcP of this invention, agonistic and antagonisticantibodies to IL-1R AcP are useful as therapeutic agents in thetreatment and prevention of diseases associated with brain and CNStissue.

[0125] The IL-1R AcP polypeptide and active fragments of the IL-1R AcPpolypeptide of this invention are particularly useful as targets inassays designed to screen for and discover inhibitors or enhancers ofsignaling pathways of IL-1 and IL-1 receptor family members. IL-1 familymembers that bind to an IL-1 receptor or IL-1 receptor family memberinteract with an IL-1R AcP of this invention to initiate a signalingresponse that includes the association or interaction of the cytoplasmicdomains of the IL-1 receptor or receptor family member and IL-1R AcPwith MyD88 and signaling through IRAK-1, IRAK-2, IRAK-M, or TRAF6. Thus,the disclosed IL-1R AcP polypeptides find utility in in vitro screeningassays directed toward the discovery of agents that antagonize ofagonize the interaction of the IL-1R AcP cytoplasmic domain with MyD88,IRAK-1, IRAK-2, IRAK-M and TRAF6. Similarly, the disclosed IL-1R AcPpolypeptides find utility in screening assays directed toward thediscovery of agents that interrupt or enhance the association of IL-1family members and/or IL-1 receptor family member or IL-1 receptor.

[0126] Advantageously, inhibitors that specifically disrupt, forexample, the association of IL-1R AcP with one or more IL-1 familymember or IL-1 receptor family members, or inhibitors that disruptintracellular signaling factors are not likely to effect signaling fromother cytokine receptors and thus can be useful therapeutic agents.Since, in some situations, as described above, the IL-1R AcP of thisinvention may antagonize the cellular responses to IL-1, or IL-1 familymembers, agonists of the inhibitory capacity of the IL-1R AcP of thisinvention are useful immunosuppressants or anti-inflammatory agents.Conversely, therapeutic agents that suppress or inhibit a negativeregulatory or inhibitory function of IL-1R AcP are useful immunoupregulators, e.g. provide increased immune activity and enhance theeffects of one or more IL-1 family members in individuals with depressedfunction. In situations where IL-1R AcP of this invention acts in apro-inflammatory capacity, by activating NF-κB, therapeutic agents thatare antagonists of IL-1R AcP of this invention are useful as ananti-inflammatory agent. Such agents are useful to treat inflammatoryconditions described herein. Accordingly, IL-1 family memberimmunoregulators, that are discovered using the disclosed IL-1R AcPpolypeptides are useful as therapeutics in the treatment of diseasesmediated by IL-1 family members as discussed below.

[0127] Suitable screening methods of this invention include methods thatassay test compounds for their ability to modulate IL-1R AcPinteractions with intracellular signaling factors and their ability tomodulate activities mediated by such IL-1R AcP interactions. To thisend, the present invention includes compounds that modulate IL-1R AcPinteractions and that are identified by the screening methods of thepresent invention. In general, screening methods of this inventioninvolve allowing the disclosed IL-1R AcP polypeptide or IL-1R AcPpolypeptide fragment, e.g. the cytoplasmic domain or fragment thereof,that is known to bind or interact with an intracellular signalingfactor, e.g. MyD88, to interact with the factor under conditions inwhich the IL-1R AcP polypeptide is known to bind or interact with thefactor. The IL-1R AcP interaction is allowed to occur in the presence ofa test compound or the test compound is allowed to contact the IL-1R AcPsubsequent to their interaction. By observing the effect that the testcompound has on the known binding characteristics of the IL-1R AcPpolypeptide or fragment, test compounds that enhance IL-1R AcPinteractions can be determined and test compounds that inhibit IL-1R AcPinteractions can be identified.

[0128] Typical test compounds are small molecules or peptides and may bepart of extensive small molecule libraries developed for use inscreening methods. IL-1R AcP polypeptides that may be used in screeningmethods include the full length herein disclosed IL-1R AcP, thecytoplasmic domain of the disclosed IL-1R AcP polypeptide, fragments ofthe cytoplasmic domain that interact with signal transduction factors,and polypeptide mutants or analogs or variants that interact signaltransduction factors. Particularly useful signal transduction factorsinclude MyD88 and down stream factors such as IRAK-1, IRAK-2, IRAK-M andTRAF6. The signal transduction factors may be recombinantly prepared andused directly in cells or isolated. Similarly, native signaltransduction factors can be used in cells or isolated and used in invitro assays.

[0129] Specific screening methods are known in the art and many areextensively incorporated in high throughput test systems so that largenumbers of test compounds can be screened within a short amount of time.Suitable screening methods can be performed in a variety of formatsincluding, but not limited to, binding assay screens, functional assayscreens and cell based screens. By observing the affect that testcompounds have on the signal transduction factor interaction with IL-1RAcP in binding assays, on activity mediated by IL-1 family members infunctional tests and in cell based screens, compounds that are potentialtherapeutics because they can modulate the interaction of IL-1R AcP withsignal transduction factors and IL-1 family members and thus the effectsof are identified.

[0130] Binding assays and their use in screening methodologies are knownin the art. For example, U.S. Pat. No. 5,767,244 (incorporated herein byreference) describes methods useful for screening compounds that areactive at the level of a TRAF6 modulatable cellular function. Inparticular, binding assays can be used to screen for test compounds thatare capable of modulating binding functions. Suitable assays includestandard protein-protein interaction tests that demonstrate the presenceor absence of protein-protein interactions and measure bindingaffinities. Typically, such binding assays involve incubating a testmixture under conditions in which the desired signal transduction factorand polypeptide, polypeptide fragment or polypeptide analog binds with aknown binding affinity. Forms of IL-1R AcP of this invention that areparticularly useful in screening for modulators of the interactioninclude the full length IL-1R AcP cytoplasmic domain of SEQ ID NO: 2 andSEQ ID NO: 4 as disclosed above and fragments of the cytoplasmic domainthat are capable of interacting with or modulating the relevant signaltransduction factor.

[0131] Protein-protein interactions can be observed and measured inbinding assays using a variety of detection methodologies that include,but are not limited to, surface plasmon resonance (Biacore), radioimmunebased assays, and fluorescence polarization binding assays. Whenperformed in the presence of a test compound, the ability of the testcompound to modulate (e.g. inhibit or enhance) the protein-proteinbinding affinity is measured. Test compounds shown to modulate IL-1R AcPand IL-1 family member interactions may be therapeutic agents fordiseases associated with IL-1 family members.

[0132] The disclosed IL-1R AcP polypeptide, fragments (including thecytoplasmic domain fragments identified above), mutants and analogs arealso useful in cell based assay methods that screen for test compoundswhich are inhibitors or modulators of the IL-1R AcP/signal transductionfactor interactions. Advantageously, cell based assays are mechanismbased and can be designed to assay test compounds for their cellmembrane permeability characteristics; their ability to modulaterelevant interactions; their ability to selectivity modulate a specificactivity mediated by IL-1 family members; and their cell toxicitycharacteristics. A number of cell based methods are known in the art.Many of the assays are based upon a yeast two-hybrid assay or mammaliantwo-hybrid assay. (See White, Proc. Natl. Acad. Sci. USA 93:10001-10003,1996). Yeast two hybrid assays as they relate to selecting smallmolecule inhibitors of protein-protein interactions are described inHuang et al. Proc. Natl. Acad. Sci 94:13396-13401, 1997. Typically,these assays involve expressing proteins (e.g. the disclosed IL-1R AcPpolypeptides or suitable fragments as disclosed herein and a signaltransduction factor) whose interaction triggers a reporter gene. Testcompounds that are cell permeable can be identified for their ability tomodulate the IL-1R AcP/signal transduction factor interaction as notedby a difference in the reporter gene triggering as compared with thereporter gene triggering in the absence of the test compound.

[0133] Additional assays that are useful for discovering modulators ofIL-1R AcP interactions include in vivo functional assays. For example,test compounds can be screened for their ability to inhibit cellfunctions that are linked to diseases mediated by IL-1 family members.Such cell functions include, but are not limited to, activation orinhibition of cell specific responses, proliferation, and inflammatoryreactions based on changes in signal transduction, activating vascularendothelial cells and lymphocytes, induction of inflammatory cytokines,acute phase proteins, hematopoiesis, fever, bone resorption,prostaglandins, metalloproteinases, and adhesion molecules.

[0134] In addition to screening methodologies, IL-1R AcP polypeptidesdisclosed herein are useful for structure-based design of inhibitors ofpathways and diseases mediated by IL-1 family members. Suchstructure-based design is also known as “rational drug design.” TheIL-1R AcP polypeptides, IL-1R AcP complexes with IL-1 receptor familymembers can be three-dimensionally analyzed by, for example, X-raycrystallography, nuclear magnetic resonance or homology modeling, all ofwhich are well-known methods. The use of IL-1R AcP structuralinformation in molecular modeling software systems to assist ininhibitor design is also encompassed by the invention. Suchcomputer-assisted modeling and drug design may utilize information suchas chemical conformational analysis, electrostatic potential of themolecules, protein folding, etc. A particular method of the inventioncomprises analyzing the three dimensional structure of IL-1R AcP forlikely binding sites, extracellular or intracellular, of substrates,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described above.

[0135] Delivery Agents

[0136] The polypeptides described herein can be used to deliverdiagnostic or therapeutic agents to such cells or cell types found toexpress molecules with which the polypeptides complex or bind in invitro or in vivo procedures. Therefore, the polypeptides describedherein can be attached to a toxin to bind to cells that express IL-1receptor family members to which they bind and specifically kill thesecells. The methodology can be similar to the successful use of ananti-CD72 immunotoxin to treat therapy-refractory B-lineage acutelymphoblastic leukemia in SCID mice (Meyers et al., Leuk. and Lymph.18:119-122).

[0137] Detectable (diagnostic) and therapeutic agents that may beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a colorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0138] Such agents may be attached to the polypeptide by any suitableconventional procedure. One such procedure involves reacting reactivefunctional groups on an agent with functional groups on one or morepolypeptide amino acid side chains. If suitable functional groups arenot present or not present in the desired location, the protein or agentmay be derivatized to generate or attach a desired reactive functionalgroup. The derivatization may involve attachment of one of thebifunctional coupling reagents available for attaching various moleculesto proteins (Pierce Chemical Company, Rockford, Ill.). A number oftechniques for radiolabeling proteins are known. Radionuclide metals maybe attached to polypeptides by using a suitable bifunctional chelatingagent, for example. The foregoing described procedures can beapplication to preparing conjugates of polypeptide of this invention anda suitable diagnostic or therapeutic agent (preferably covalentlylinked). Such conjugates are administered or otherwise employed in anamount appropriate for the particular application.

[0139] Further embodiments of the present invention include therapeuticuses of the disclosed IL-1R AcP polypeptides and polypeptide fragments.Such therapeutic uses generally involve the inhibition of the action ofIL-1 or IL-1 homologues that play a central role in protection againstinfection and immune inflammatory responses. IL-1 is involved incellular signal transduction, activating vascular endothelial cells andlymphocytes, induction of inflammatory cytokines, acute phase proteins,hematopoiesis, fever, bone resorption, prostaglandins,metalloproteinases, and adhesion molecules. IL-1R AcP described hereinis involved in the functions noted above as well as modulatinginflammatory responses and therefore perhaps be involved in the negativeregulation of and the causation and maintenance of inflammatory and/orautoimmune diseases such as rheumatoid arthritis, inflammatory boweldisease, and psoriasis. As such, alterations in the expression and/oractivation of IL-1R AcP can have profound effects on a plethora ofcellular processes, including, but not limited to, activation orinhibition of cell specific responses, proliferation, and inflammatoryreactions based on changes in signal transduction.

[0140] For many IL-1 family members, the cellular signaling involves amolecular activation cascade, during which a receptor propagates aligand-receptor mediated signal by specifically activating intracellularkinases which phosphorylate target substrates, resulting in theactivation of the transcription factors NFkB and AP1, the proteinkinases Jun N-terminal kinase and p38 map kinase, the enzymes COX-2leading to prostaglandin production and iNOS leading to nitric oxideproduction, and inflammation in general.

[0141] Thus, isolated IL-1R AcP polypeptides described herein, whichinteract with at least one IL-1 family member and IL-1 receptor familymember, can be useful as therapeutic agents. In situations where anIL-1R AcP of this invention has an inhibitory role, polypeptide andpolypeptide fragments of this invention can be useful in inhibitingsignaling. In cases in which IL-1R AcP of this invention is not anegative regulator, soluble polypeptides can interact with relevant IL-1receptor family members or IL-1 family members, and inhibit theactivation of cells through cell-associated IL-1R AcP. Furthermore, thecytoplasmic domain or fragments thereof that are capable of bindingsignal transduction factors may be engineered for introduction into theintracellular environment where they bind to signal transduction factorsand inhibit signaling through cell-associated IL-1R AcP.

[0142] Specific medical conditions and diseases that are treatable orpreventable with the IL-1R AcP polypeptides of this invention includecoeliac disease, Crohn's disease; ulcerative colitis; idiopathicgastroparesis; pancreatitis, including chronic pancreatitis;inflammatory bowel disease and ulcers, including gastric and duodenalulcers.

[0143] As described above and shown in Example 1, IL-1R AcP of thisinvention is expressed in brain/CNS tissue. Thus, modulators of IL-1RAcP activity that are described above are useful in treating diseasesassociated with the central nervous system, the head, the spinal chordand brain, including head and spinal chord injuries and subduralhematoma due to trauma to the head. The therapeutic agents describedherein and discovered through screening activities can be used to treatcranial neurologic damage and to prevent and treat cervicogenicheadaches and to treat neurological side effects associated with brainirradiation.

[0144] Therapeutic agents that are modulators of IL-1R AcP of thisinvention can also be used to treat or prevent primary amyloidosis. Inaddition, secondary amyloidosis that is characteristic of variousconditions also are treatable modulators of IL-1R AcP of this invention.Such conditions include: Alzheimer's disease, secondary reactiveamyloidosis; Down's syndrome; and dialysis-associated amyloidosis.Various other medical disorders treatable with the therapeutic agentsthat are modulators of IL-1R AcP include; multiple sclerosis; Behcet'ssyndrome; Sjogren's syndrome; autoimmune hemolytic anemia; betathalassemia; amyotrophic lateral sclerosis (Lou Gehrig's Disease);Parkinson's disease; and tenosynovitis of unknown cause, as well asvarious autoimmune disorders or diseases associated with hereditarydeficiencies, including x-linked mental retardation.

[0145] Further uses for the therapeutic agents that are modulators ofthe IL-1R AcP described here include treating central nervous system(CNS) injuries, including the effects of neurotoxic neurotransmittersdischarged during excitation of inflammation in the central nervoussystem and to inhibit or prevent the development of glial scars at sitesof central nervous system injury. In connection with central nervoussystem medical conditions, the modulators of IL-1R AcP of this inventionare useful in treating temporal lobe epilepsy. In connection withepilepsy and the treatment of seizures, the modulators of IL-1R AcP mayreduce the severity and number of recurring seizures, and reduce theseverity of the deleterious effects of seizures. The therapeuticmodulators are also useful for reducing neuronal loss, neuronaldegeneration, and gliosis associated with seizures.

[0146] Further, consistent with its presence in brain and CNS tissue,modulators of IL-1R AcP described herein are useful for treatingcritical illness polyneuropathy and myopathy (CIPNM) acutepolyneuropathy; anorexia nervosa; Bell's palsy; chronic fatiguesyndrome; transmissible dementia, including Creutzfeld-Jacob disease;demyelinating neuropathy; Guillain-Barre syndrome; vertebral discdisease; Gulf war syndrome; chronic inflammatory demyelinatingpolyneuropathy, myasthenia gravis; silent cerebral ischemia; sleepdisorders, including narcolepsy and sleep apnea; chronic neuronaldegeneration; and stroke, including cerebral ischemic diseases.

[0147] Rheumatic disorders that are treatable with the polypeptides ofthis invention include adult and juvenile rheumatoid arthritis;scleroderma; systemic lupus erythematosus; gout; osteoarthritis;polymyalgia rheumatica; seronegative spondylarthropathies, includingankylosing spondylitis, and Reiter's disease, psoriatic arthritis andchronic Lyme arthritis. Also treatable or preventable with thesepolypeptides are Still's disease and uveitis associated with rheumatoidarthritis. In addition, the polypeptide therapies of the invention areused in treating disorders resulting in inflammation of the voluntarymuscle and other muscles, including dermatomyositis, inclusion bodymyositis, polymyositis, and lymphangioleimyomatosis.

[0148] The methods described herein can be treated with the polypeptidesand inhibitors of this invention in combination with other cytokines,cytokine inhibitors and reagents. For example, IL-18 antagonists;including soluble IL-18 receptor, antibodies to IL-18 or the IL-18receptor, IL-18 binding protein; TNF inhibitors, including ENBREL®; IL-1inhibitors, including soluble forms of type II IL-1R, type II IL-1R,antibodies to IL-1, antibodies to type I IL-1R; and or other activeagents that are effective in treating the disclosed medical conditionsand diseases.

[0149] Polypeptides can be introduced into the extracellular environmentby well-known means, such as by administering the protein intravenouslyor coupling it to a monoclonal antibody targeted to a specific celltype, to thereby affect signaling. When used as a therapeutic agent,polypeptides of the invention can be formulated into pharmaceuticalcompositions according to known methods. The polypeptides can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzylalcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co.In addition, such compositions can contain the polypeptides complexedwith polyethylene glycol (PEG), metal ions, or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, etc., or incorporated into liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of polypeptides of the invention.

[0150] The dosage of the composition can be readily determined by thoseof ordinary skill in the art. The amount to be administered and thefrequency of administration can be determined empirically and will takeinto consideration the age and size of the patient being treated, aswell as the malady being treated.

[0151] Treatment comprises administering the composition by any methodfamiliar to those of ordinary skill in the art, including intravenous,intraperitoneal, intracorporeal injection, intra-articular,intraventricular, intrathecal, intramuscular, subcutaneous, topically,tonsillar, intranasally, intravaginally, and orally. The composition mayalso be given locally, such as by injection into the particular area,either intramuscularly or subcutaneously.

[0152] The polypeptides described herein can also be used forstructure-based design of IL-1 inhibitors, or IL-1 receptor familymember inhibitors or inhibitors of IL-1R AcP function. Suchstructure-based design is also known as “rational drug design.”Polypeptides of this invention can be three dimensionally analyzed by,for example, X-ray crystallography, nuclear magnetic resonance, orhomology modeling, all of which are well known methods. The use ofstructural information in molecular modeling software systems to assistin inhibitor design and inhibitor-interaction is also encompassed by theinvention. Such computer-assisted modeling and drug design may utilizeinformation such as chemical conformational analysis, electrostaticpotential of the molecules, protein folding, etc. For example, most ofthe design of class-specific inhibitors of metalloproteases has focusedon attempts to chelate or bind the catalytic zinc atom. Syntheticinhibitors are usually designed to contain a negatively charged moietyto which is attached a series of other groups designed to fit thespecificity pockets of the particular protease. A particular method ofthe invention comprises analyzing the three dimensional structure of theIL-1R AcP of this invention for likely binding sites of substrates,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described above.

[0153] The polynucleotides and polypeptides of this invention, andantibodies against the polypeptides described herein can be used asreagents in a variety of research protocols. A sample of such researchprotocols are given in Sambrook et al. Molecular Cloning: A LaboratoryManual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, (1989). Forexample, these reagents can serve as markers for cell specific or tissuespecific expression of RNA or proteins. Similarly, these reagents can beused to investigate constituitive and transient expression of IL-1R AcPsplice variant RNA or polypeptides.

[0154] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as immunogensin producing antibodies immunoreactive therewith.

[0155] The IL-1R AcP splice variant, fragments of the splice variant,the cytoplasmic domain, fragments of the cytomplasmic domain, aminoacids 379-687, amino acids 384-687, and amino acids 449-687, all of SEQID NO: 2 and amino acids 449-685, amino acids 379-687, amino acids389-687, all of SEQ ID NO: 4 can be utilized to prepare antibodies thatspecifically bind to IL-1R AcP. The term “antibodies” is meant toinclude polyclonal antibodies, monoclonal antibodies, fragments thereof,such as F(ab′)2 and Fab fragments, as well as any recombinantly producedbinding partners. Antibodies are defined to be specifically binding ifthey bind the IL-1R AcP polypeptide with a K_(a) of greater than orequal to about 10⁷ M⁻¹. Affinities of binding partners or antibodies canbe readily determined using conventional techniques, for example thosedescribed by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

[0156] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well known in the art.In general, purified peptide that is appropriately conjugated isadministered to the host animal typically through parenteral injection.The immunogenicity of the polypeptide can be enhanced through the use ofan adjuvant, for example, Freund's complete or incomplete adjuvant.Following booster immunizations, small samples of serum are collectedand tested for reactivity to the polypeptide. Examples of various assaysuseful for such determination include those described in Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; as well as procedures, such as countercurrentimmuno-electrophoresis (CIEP), radioimmnunoassay,radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA),dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and4,486,530.

[0157] Monoclonal antibodies can be readily prepared using well knownprocedures. See, for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980. Briefly, the host animals,such as mice, are injected intraperitoneally at least once andpreferably at least twice at about 3 week intervals with isolated andpurified peptide, optionally in the presence of adjuvant. Mouse sera arethen assayed by conventional dot blot technique or antibody capture(ABC) to determine which animal is best to fuse. Approximately two tothree weeks later, the mice are given an intravenous boost of peptide.Mice are later sacrificed and spleen cells fused with commerciallyavailable myeloma cells, such as Ag8.653 (ATCC), following establishedprotocols. Briefly, the myeloma cells are washed several times in mediaand fused to mouse spleen cells at a ratio of about three spleen cellsto one myeloma cell. The fusing agent can be any suitable agent used inthe art, for example, polyethylene glycol (PEG). Fusion is plated outinto plates containing media that allows for the selective growth of thefused cells. The fused cells can then be allowed to grow forapproximately eight days. Supernatants from resultant hybridomas arecollected and added to a plate that is first coated with goat anti-mouseIg. Following washes, a label, such as ¹²⁵1-IL-1R AcP, is added to eachwell followed by incubation. Positive wells can be subsequently detectedby autoradiography. Positive clones can be grown in bulk culture andsupernatants are subsequently purified over a Protein A column(Pharmacia).

[0158] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7:394 (1989).

[0159] Antigen-binding fragments of such antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0160] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques, and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14: 139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0161] In one embodiment, the antibodies are specific for thepolypeptides of the present invention, and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0162] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0163] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0164] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

[0165] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0166] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet TABLE I IL-1R AcP of IL-1R AcP of BRAIN TISSUE IL-1R WO96/23067 this invention Total Brain ++ ++ ++ Fetal brain ++ ++ ++Frontal lobe ++ + ++ Temporal lobe ++ ++ ++ Occipital lobe ++ ++ ++Parietal lobe ++ ++ ++ Cerebral cortex ++ ++ ++ Pons ++ ++ ++ Cerebellum++ ++ ++ Medulla ++ ++ ++ Oblongata Hippocampus ++ ++ ++ Amygdala * ++++ Thalamus ++ ++ ++ Corpus Callosum + −− ++ Cerebral ++ ++ ++ PedunclesSpinal cord ++ ++ ++ Substantia nigra ++ ++ ++

[0167] The embodiments within the specification provide an illustrationof embodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan recognizes many otherembodiments are encompassed by the claimed invention.

1 4 1 2064 DNA Homo sapiens CDS (1)..(2064) 1 atg aca ctt ctg tgg tgtgta gtg agt ctc tac ttt tat gga atc ctg 48 Met Thr Leu Leu Trp Cys ValVal Ser Leu Tyr Phe Tyr Gly Ile Leu 1 5 10 15 caa agt gat gcc tca gaacgc tgc gat gac tgg gga cta gac acc atg 96 Gln Ser Asp Ala Ser Glu ArgCys Asp Asp Trp Gly Leu Asp Thr Met 20 25 30 agg caa atc caa gtg ttt gaagat gag cca gct cgc atc aag tgc cca 144 Arg Gln Ile Gln Val Phe Glu AspGlu Pro Ala Arg Ile Lys Cys Pro 35 40 45 ctc ttt gaa cac ttc ttg aaa ttcaac tac agc aca gcc cat tca gct 192 Leu Phe Glu His Phe Leu Lys Phe AsnTyr Ser Thr Ala His Ser Ala 50 55 60 ggc ctt act ctg atc tgg tat tgg actagg cag gac cgg gac ctt gag 240 Gly Leu Thr Leu Ile Trp Tyr Trp Thr ArgGln Asp Arg Asp Leu Glu 65 70 75 80 gag cca att aac ttc cgc ctc ccc gagaac cgc att agt aag gag aaa 288 Glu Pro Ile Asn Phe Arg Leu Pro Glu AsnArg Ile Ser Lys Glu Lys 85 90 95 gat gtg ctg tgg ttc cgg ccc act ctc ctcaat gac act ggc aac tat 336 Asp Val Leu Trp Phe Arg Pro Thr Leu Leu AsnAsp Thr Gly Asn Tyr 100 105 110 acc tgc atg tta agg aac act aca tat tgcagc aaa gtt gca ttt ccc 384 Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys SerLys Val Ala Phe Pro 115 120 125 ttg gaa gtt gtt caa aaa gac agc tgt ttcaat tcc ccc atg aaa ctc 432 Leu Glu Val Val Gln Lys Asp Ser Cys Phe AsnSer Pro Met Lys Leu 130 135 140 cca gtg cat aaa ctg tat ata gaa tat ggcatt cag agg atc act tgt 480 Pro Val His Lys Leu Tyr Ile Glu Tyr Gly IleGln Arg Ile Thr Cys 145 150 155 160 cca aat gta gat gga tat ttt cct tccagt gtc aaa ccg act atc act 528 Pro Asn Val Asp Gly Tyr Phe Pro Ser SerVal Lys Pro Thr Ile Thr 165 170 175 tgg tat atg ggc tgt tat aaa ata cagaat ttt aat aat gta ata ccc 576 Trp Tyr Met Gly Cys Tyr Lys Ile Gln AsnPhe Asn Asn Val Ile Pro 180 185 190 gaa ggt atg aac ttg agt ttc ctc attgcc tta att tca aat aat gga 624 Glu Gly Met Asn Leu Ser Phe Leu Ile AlaLeu Ile Ser Asn Asn Gly 195 200 205 aat tac aca tgt gtt gtt aca tat ccagaa aat gga cgt acg ttt cat 672 Asn Tyr Thr Cys Val Val Thr Tyr Pro GluAsn Gly Arg Thr Phe His 210 215 220 ctc acc agg act ctg act gta aag gtagta ggc tct cca aaa aat gca 720 Leu Thr Arg Thr Leu Thr Val Lys Val ValGly Ser Pro Lys Asn Ala 225 230 235 240 gtg ccc cct gtg atc cat tca cctaat gat cat gtg gtc tat gag aaa 768 Val Pro Pro Val Ile His Ser Pro AsnAsp His Val Val Tyr Glu Lys 245 250 255 gaa cca gga gag gag cta ctc attccc tgt acg gtc tat ttt agt ttt 816 Glu Pro Gly Glu Glu Leu Leu Ile ProCys Thr Val Tyr Phe Ser Phe 260 265 270 ctg atg gat tct cgc aat gag gtttgg tgg acc att gat gga aaa aaa 864 Leu Met Asp Ser Arg Asn Glu Val TrpTrp Thr Ile Asp Gly Lys Lys 275 280 285 cct gat gac atc act att gat gtcacc att aac gaa agt ata agt cat 912 Pro Asp Asp Ile Thr Ile Asp Val ThrIle Asn Glu Ser Ile Ser His 290 295 300 agt aga aca gaa gat gaa aca agaact cag att ttg agc atc aag aaa 960 Ser Arg Thr Glu Asp Glu Thr Arg ThrGln Ile Leu Ser Ile Lys Lys 305 310 315 320 gtt acc tct gag gat ctc aagcgc agc tat gtc tgt cat gct aga agt 1008 Val Thr Ser Glu Asp Leu Lys ArgSer Tyr Val Cys His Ala Arg Ser 325 330 335 gcc aaa ggc gaa gtt gcc aaagca gcc aag gtg aag cag aaa gtg cca 1056 Ala Lys Gly Glu Val Ala Lys AlaAla Lys Val Lys Gln Lys Val Pro 340 345 350 gct cca aga tac aca gtg gaactg gct tgt ggt ttt gga gcc aca gtc 1104 Ala Pro Arg Tyr Thr Val Glu LeuAla Cys Gly Phe Gly Ala Thr Val 355 360 365 ctg cta gtg gtg att ctc attgtt gtt tac cat gtt tac tgg cta gag 1152 Leu Leu Val Val Ile Leu Ile ValVal Tyr His Val Tyr Trp Leu Glu 370 375 380 atg gtc cta ttt tac cgg gctcat ttt gga aca gat gaa acc att tta 1200 Met Val Leu Phe Tyr Arg Ala HisPhe Gly Thr Asp Glu Thr Ile Leu 385 390 395 400 gat gga aaa gag tat gatatt tat gta tcc tat gca agg aat gcg gaa 1248 Asp Gly Lys Glu Tyr Asp IleTyr Val Ser Tyr Ala Arg Asn Ala Glu 405 410 415 gaa gaa gaa ttt gta ttactg acc ctc cgt gga gtt ttg gag aat gaa 1296 Glu Glu Glu Phe Val Leu LeuThr Leu Arg Gly Val Leu Glu Asn Glu 420 425 430 ttt gga tac aag ctg tgcatc ttt gac cga gac agt ctg cct ggg gga 1344 Phe Gly Tyr Lys Leu Cys IlePhe Asp Arg Asp Ser Leu Pro Gly Gly 435 440 445 aat aca gtg gaa gca gttttt gat ttc att cag aga agc aga agg atg 1392 Asn Thr Val Glu Ala Val PheAsp Phe Ile Gln Arg Ser Arg Arg Met 450 455 460 att gtt gtt ctg agc cctgac tat gtg aca gaa aag agc atc agc atg 1440 Ile Val Val Leu Ser Pro AspTyr Val Thr Glu Lys Ser Ile Ser Met 465 470 475 480 ctg gag ttt aaa ctgggt gtc atg tgc cag aac tcc att gcc acc aag 1488 Leu Glu Phe Lys Leu GlyVal Met Cys Gln Asn Ser Ile Ala Thr Lys 485 490 495 ctc att gtg gtt gagtac cgt ccc ctt gag cac ccg cac cca ggc att 1536 Leu Ile Val Val Glu TyrArg Pro Leu Glu His Pro His Pro Gly Ile 500 505 510 ctt cag ctc aaa gagtct gtg tct ttt gtg agc tgg aag gga gaa aag 1584 Leu Gln Leu Lys Glu SerVal Ser Phe Val Ser Trp Lys Gly Glu Lys 515 520 525 tcc aaa cat tct ggctct aaa ttc tgg aaa gct ttg cgg ttg gct ctt 1632 Ser Lys His Ser Gly SerLys Phe Trp Lys Ala Leu Arg Leu Ala Leu 530 535 540 ccc ctg aga agt ctgagt gcc agt tct ggc tgg aat gag agc tgc tct 1680 Pro Leu Arg Ser Leu SerAla Ser Ser Gly Trp Asn Glu Ser Cys Ser 545 550 555 560 tcc cag tct gacatc agt ctg gat cac gtt caa agg agg aga agt cgt 1728 Ser Gln Ser Asp IleSer Leu Asp His Val Gln Arg Arg Arg Ser Arg 565 570 575 ttg aaa gag ccccca gaa ctt cag agc tca gag agg gct gca ggt agc 1776 Leu Lys Glu Pro ProGlu Leu Gln Ser Ser Glu Arg Ala Ala Gly Ser 580 585 590 cct cca gcc ccaggc nca atg tcc aag cac cga ggg aag tcc tcc gcc 1824 Pro Pro Ala Pro GlyXaa Met Ser Lys His Arg Gly Lys Ser Ser Ala 595 600 605 acc tgc cgc tgttgt gtc acc tac tgt gaa gga gag aat cac ctt agg 1872 Thr Cys Arg Cys CysVal Thr Tyr Cys Glu Gly Glu Asn His Leu Arg 610 615 620 aac aag agc cgggca gag att cat aac cag ccc cag tgg gag aca cac 1920 Asn Lys Ser Arg AlaGlu Ile His Asn Gln Pro Gln Trp Glu Thr His 625 630 635 640 ctc tgt aagcct gtt ccc caa gag tca gaa act caa tgg ata caa aat 1968 Leu Cys Lys ProVal Pro Gln Glu Ser Glu Thr Gln Trp Ile Gln Asn 645 650 655 ggc acc agattg gaa ccc cct gct ccc cag atc tca gcc ctt gct ctt 2016 Gly Thr Arg LeuGlu Pro Pro Ala Pro Gln Ile Ser Ala Leu Ala Leu 660 665 670 cat cat ttcacg gac tta tcc aat aac aac gac ttt tat atc cta taa 2064 His His Phe ThrAsp Leu Ser Asn Asn Asn Asp Phe Tyr Ile Leu 675 680 685 2 687 PRT Homosapiens misc_feature (598)..(598) The ′Xaa′ at location 598 stands forThr or Pro. 2 Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr GlyIle Leu 1 5 10 15 Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly LeuAsp Thr Met 20 25 30 Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg IleLys Cys Pro 35 40 45 Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr AlaHis Ser Ala 50 55 60 Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp ArgAsp Leu Glu 65 70 75 80 Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg IleSer Lys Glu Lys 85 90 95 Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn AspThr Gly Asn Tyr 100 105 110 Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys SerLys Val Ala Phe Pro 115 120 125 Leu Glu Val Val Gln Lys Asp Ser Cys PheAsn Ser Pro Met Lys Leu 130 135 140 Pro Val His Lys Leu Tyr Ile Glu TyrGly Ile Gln Arg Ile Thr Cys 145 150 155 160 Pro Asn Val Asp Gly Tyr PhePro Ser Ser Val Lys Pro Thr Ile Thr 165 170 175 Trp Tyr Met Gly Cys TyrLys Ile Gln Asn Phe Asn Asn Val Ile Pro 180 185 190 Glu Gly Met Asn LeuSer Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly 195 200 205 Asn Tyr Thr CysVal Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His 210 215 220 Leu Thr ArgThr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala 225 230 235 240 ValPro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys 245 250 255Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe 260 265270 Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys 275280 285 Pro Asp Asp Ile Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser His290 295 300 Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile LysLys 305 310 315 320 Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys HisAla Arg Ser 325 330 335 Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val LysGln Lys Val Pro 340 345 350 Ala Pro Arg Tyr Thr Val Glu Leu Ala Cys GlyPhe Gly Ala Thr Val 355 360 365 Leu Leu Val Val Ile Leu Ile Val Val TyrHis Val Tyr Trp Leu Glu 370 375 380 Met Val Leu Phe Tyr Arg Ala His PheGly Thr Asp Glu Thr Ile Leu 385 390 395 400 Asp Gly Lys Glu Tyr Asp IleTyr Val Ser Tyr Ala Arg Asn Ala Glu 405 410 415 Glu Glu Glu Phe Val LeuLeu Thr Leu Arg Gly Val Leu Glu Asn Glu 420 425 430 Phe Gly Tyr Lys LeuCys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly 435 440 445 Asn Thr Val GluAla Val Phe Asp Phe Ile Gln Arg Ser Arg Arg Met 450 455 460 Ile Val ValLeu Ser Pro Asp Tyr Val Thr Glu Lys Ser Ile Ser Met 465 470 475 480 LeuGlu Phe Lys Leu Gly Val Met Cys Gln Asn Ser Ile Ala Thr Lys 485 490 495Leu Ile Val Val Glu Tyr Arg Pro Leu Glu His Pro His Pro Gly Ile 500 505510 Leu Gln Leu Lys Glu Ser Val Ser Phe Val Ser Trp Lys Gly Glu Lys 515520 525 Ser Lys His Ser Gly Ser Lys Phe Trp Lys Ala Leu Arg Leu Ala Leu530 535 540 Pro Leu Arg Ser Leu Ser Ala Ser Ser Gly Trp Asn Glu Ser CysSer 545 550 555 560 Ser Gln Ser Asp Ile Ser Leu Asp His Val Gln Arg ArgArg Ser Arg 565 570 575 Leu Lys Glu Pro Pro Glu Leu Gln Ser Ser Glu ArgAla Ala Gly Ser 580 585 590 Pro Pro Ala Pro Gly Xaa Met Ser Lys His ArgGly Lys Ser Ser Ala 595 600 605 Thr Cys Arg Cys Cys Val Thr Tyr Cys GluGly Glu Asn His Leu Arg 610 615 620 Asn Lys Ser Arg Ala Glu Ile His AsnGln Pro Gln Trp Glu Thr His 625 630 635 640 Leu Cys Lys Pro Val Pro GlnGlu Ser Glu Thr Gln Trp Ile Gln Asn 645 650 655 Gly Thr Arg Leu Glu ProPro Ala Pro Gln Ile Ser Ala Leu Ala Leu 660 665 670 His His Phe Thr AspLeu Ser Asn Asn Asn Asp Phe Tyr Ile Leu 675 680 685 3 2058 DNA Musmusculus CDS (1)..(2058) 3 atg gga ctt ctg tgg tat ttg atg agt ctg tccttc tat ggg atc ctg 48 Met Gly Leu Leu Trp Tyr Leu Met Ser Leu Ser PheTyr Gly Ile Leu 1 5 10 15 cag agt cat gct tcg gag cgc tgt gat gac tgggga cta gat acc atg 96 Gln Ser His Ala Ser Glu Arg Cys Asp Asp Trp GlyLeu Asp Thr Met 20 25 30 cga caa atc caa gtg ttt gaa gat gag ccg gct cgaatc aag tgc ccc 144 Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg IleLys Cys Pro 35 40 45 ctc ttt gaa cac ttc ctg aag tac aac tac agc act gcccat tcc tct 192 Leu Phe Glu His Phe Leu Lys Tyr Asn Tyr Ser Thr Ala HisSer Ser 50 55 60 ggc ctt acc ctg atc tgg tac tgg acc agg caa gac cgg gacctg gag 240 Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp LeuGlu 65 70 75 80 gag ccc att aac ttc cgc ctc cca gag aat cgc atc agt aaggag aaa 288 Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys GluLys 85 90 95 gat gtg ctc tgg ttc cgg ccc acc ctc ctc aat gac acg ggc aattac 336 Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr100 105 110 acc tgc atg ttg agg aac aca act tac tgc agc aaa gtt gca tttccc 384 Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro115 120 125 ctg gaa gtt gtt cag aag gac agc tgt ttc aat tct gcc atg agattc 432 Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Ala Met Arg Phe130 135 140 cca gtg cac aag atg tat att gaa cat ggc att cat aag atc acatgt 480 Pro Val His Lys Met Tyr Ile Glu His Gly Ile His Lys Ile Thr Cys145 150 155 160 cca aat gta gac gga tac ttt cct tcc agt gtc aaa cca tcggtc act 528 Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser ValThr 165 170 175 tgg tat aag ggt tgt act gaa ata gtg gac ttt cat aat gtacta ccc 576 Trp Tyr Lys Gly Cys Thr Glu Ile Val Asp Phe His Asn Val LeuPro 180 185 190 gag ggc atg aac ttg agc ttt ttc atc ccc ttg gtt tca aataac gga 624 Glu Gly Met Asn Leu Ser Phe Phe Ile Pro Leu Val Ser Asn AsnGly 195 200 205 aat tac aca tgt gtg gtt aca tat cct gaa aac gga cgt ctcttt cac 672 Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Leu PheHis 210 215 220 ctc acc agg act gtg act gta aag gtg gtg ggc tca cca aaggat gca 720 Leu Thr Arg Thr Val Thr Val Lys Val Val Gly Ser Pro Lys AspAla 225 230 235 240 ttg cca ccc cag atc tat tct cca aat gac cgt gtt gtctat gag aaa 768 Leu Pro Pro Gln Ile Tyr Ser Pro Asn Asp Arg Val Val TyrGlu Lys 245 250 255 gaa cca gga gag gaa ctg gtt att ccc tgc aaa gtc tatttc agt ttc 816 Glu Pro Gly Glu Glu Leu Val Ile Pro Cys Lys Val Tyr PheSer Phe 260 265 270 att atg gac tcc cac aat gag gtc tgg tgg acc att gatgga aag aag 864 Ile Met Asp Ser His Asn Glu Val Trp Trp Thr Ile Asp GlyLys Lys 275 280 285 cct gat gac gtc aca gtc gac atc act att aat gaa agtgta agt tat 912 Pro Asp Asp Val Thr Val Asp Ile Thr Ile Asn Glu Ser ValSer Tyr 290 295 300 tct tca acg gaa gat gaa aca agg act cag att ttg agcatc aag aaa 960 Ser Ser Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser IleLys Lys 305 310 315 320 gtc acc ccg gag gat ctc agg cgc aac tat gtc tgtcat gct cga aat 1008 Val Thr Pro Glu Asp Leu Arg Arg Asn Tyr Val Cys HisAla Arg Asn 325 330 335 acc aaa ggg gaa gct gag cag gct gcc aag gtg aaacag aaa gtc ata 1056 Thr Lys Gly Glu Ala Glu Gln Ala Ala Lys Val Lys GlnLys Val Ile 340 345 350 cca cca agg tac aca gta gaa ctc gcc tgt ggt tttgga gcc acg gtc 1104 Pro Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe GlyAla Thr Val 355 360 365 ttt ctg gta gtg gtt ctc att gtg gtt tac cat gtttac tgg ctg gag 1152 Phe Leu Val Val Val Leu Ile Val Val Tyr His Val TyrTrp Leu Glu 370 375 380 atg gtc ctc ttt tac cga gct cac ttt gga aca gatgaa aca att ctt 1200 Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp GluThr Ile Leu 385 390 395 400 gat gga aag gag tat gat att tat gtt tcc tatgca aga aat gtg gaa 1248 Asp Gly Lys Glu Tyr Asp Ile Tyr Val Ser Tyr AlaArg Asn Val Glu 405 410 415 gaa gag gaa ttt gtg ctg ctg acg ctg cgt ggagtt ttg gag aat gag 1296 Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly ValLeu Glu Asn Glu 420 425 430 ttt gga tac aag ctg tgc atc ttc gac aga gacagc ctg cct ggg gga 1344 Phe Gly Tyr Lys Leu Cys Ile Phe Asp Arg Asp SerLeu Pro Gly Gly 435 440 445 aat acc gtg gaa gca gtt ttt gat ttc att cagagg agc cga agg atg 1392 Asn Thr Val Glu Ala Val Phe Asp Phe Ile Gln ArgSer Arg Arg Met 450 455 460 att gtt gtc ctg agc cct gac tat gtg aca gaaaag agc atc agc atg 1440 Ile Val Val Leu Ser Pro Asp Tyr Val Thr Glu LysSer Ile Ser Met 465 470 475 480 ctg gag ttt aag ctg ggt gtc atg tgc cagaac tcc att gcc act aag 1488 Leu Glu Phe Lys Leu Gly Val Met Cys Gln AsnSer Ile Ala Thr Lys 485 490 495 ctc att gtg gtg gag tac cgt ccg ctt gagcaa ccc cat cca ggc atc 1536 Leu Ile Val Val Glu Tyr Arg Pro Leu Glu GlnPro His Pro Gly Ile 500 505 510 atg cag ctg aag gag tct gtg tct ttt gtaagc tgg aag gga gaa aag 1584 Met Gln Leu Lys Glu Ser Val Ser Phe Val SerTrp Lys Gly Glu Lys 515 520 525 tcc aaa cat tct ggc tcc aag ttc tgg aaggcc ttg cgt ttg gct ctt 1632 Ser Lys His Ser Gly Ser Lys Phe Trp Lys AlaLeu Arg Leu Ala Leu 530 535 540 ccc ctg aga agt ctg agc gcc agc tcc ggctgg aat gag agc tgt tct 1680 Pro Leu Arg Ser Leu Ser Ala Ser Ser Gly TrpAsn Glu Ser Cys Ser 545 550 555 560 tct cag tct gac atc agt ctg gat catgtt cag agg aga agt cgt ttg 1728 Ser Gln Ser Asp Ile Ser Leu Asp His ValGln Arg Arg Ser Arg Leu 565 570 575 aaa gag ccc cca gaa ctc cga agc tcagag agg gtg tct gga gca gag 1776 Lys Glu Pro Pro Glu Leu Arg Ser Ser GluArg Val Ser Gly Ala Glu 580 585 590 cca gcc ccg ggc acg atg tcc aag caccga ggg aaa ccc tca gca gcc 1824 Pro Ala Pro Gly Thr Met Ser Lys His ArgGly Lys Pro Ser Ala Ala 595 600 605 tgt cgc tgc tgt gtc acc tac tgt gaagga gaa agt cac ctc agg agc 1872 Cys Arg Cys Cys Val Thr Tyr Cys Glu GlyGlu Ser His Leu Arg Ser 610 615 620 aag agc cgg gca gag atg cac acg catccc cag tgg gaa aca cac ctc 1920 Lys Ser Arg Ala Glu Met His Thr His ProGln Trp Glu Thr His Leu 625 630 635 640 tgt aag cct cct ctc caa gag tctgaa agt cag tgg ata caa aat ggc 1968 Cys Lys Pro Pro Leu Gln Glu Ser GluSer Gln Trp Ile Gln Asn Gly 645 650 655 acc cga ccc gaa ccc gct ccc cagatc tca gct ctt gca ctc cgc cac 2016 Thr Arg Pro Glu Pro Ala Pro Gln IleSer Ala Leu Ala Leu Arg His 660 665 670 ttt aca gat tta tcc aat aac aatgac ttt tat atc cta taa 2058 Phe Thr Asp Leu Ser Asn Asn Asn Asp Phe TyrIle Leu 675 680 685 4 685 PRT Mus musculus 4 Met Gly Leu Leu Trp Tyr LeuMet Ser Leu Ser Phe Tyr Gly Ile Leu 1 5 10 15 Gln Ser His Ala Ser GluArg Cys Asp Asp Trp Gly Leu Asp Thr Met 20 25 30 Arg Gln Ile Gln Val PheGlu Asp Glu Pro Ala Arg Ile Lys Cys Pro 35 40 45 Leu Phe Glu His Phe LeuLys Tyr Asn Tyr Ser Thr Ala His Ser Ser 50 55 60 Gly Leu Thr Leu Ile TrpTyr Trp Thr Arg Gln Asp Arg Asp Leu Glu 65 70 75 80 Glu Pro Ile Asn PheArg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys 85 90 95 Asp Val Leu Trp PheArg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 100 105 110 Thr Cys Met LeuArg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro 115 120 125 Leu Glu ValVal Gln Lys Asp Ser Cys Phe Asn Ser Ala Met Arg Phe 130 135 140 Pro ValHis Lys Met Tyr Ile Glu His Gly Ile His Lys Ile Thr Cys 145 150 155 160Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser Val Thr 165 170175 Trp Tyr Lys Gly Cys Thr Glu Ile Val Asp Phe His Asn Val Leu Pro 180185 190 Glu Gly Met Asn Leu Ser Phe Phe Ile Pro Leu Val Ser Asn Asn Gly195 200 205 Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Leu PheHis 210 215 220 Leu Thr Arg Thr Val Thr Val Lys Val Val Gly Ser Pro LysAsp Ala 225 230 235 240 Leu Pro Pro Gln Ile Tyr Ser Pro Asn Asp Arg ValVal Tyr Glu Lys 245 250 255 Glu Pro Gly Glu Glu Leu Val Ile Pro Cys LysVal Tyr Phe Ser Phe 260 265 270 Ile Met Asp Ser His Asn Glu Val Trp TrpThr Ile Asp Gly Lys Lys 275 280 285 Pro Asp Asp Val Thr Val Asp Ile ThrIle Asn Glu Ser Val Ser Tyr 290 295 300 Ser Ser Thr Glu Asp Glu Thr ArgThr Gln Ile Leu Ser Ile Lys Lys 305 310 315 320 Val Thr Pro Glu Asp LeuArg Arg Asn Tyr Val Cys His Ala Arg Asn 325 330 335 Thr Lys Gly Glu AlaGlu Gln Ala Ala Lys Val Lys Gln Lys Val Ile 340 345 350 Pro Pro Arg TyrThr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val 355 360 365 Phe Leu ValVal Val Leu Ile Val Val Tyr His Val Tyr Trp Leu Glu 370 375 380 Met ValLeu Phe Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu 385 390 395 400Asp Gly Lys Glu Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn Val Glu 405 410415 Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu 420425 430 Phe Gly Tyr Lys Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly435 440 445 Asn Thr Val Glu Ala Val Phe Asp Phe Ile Gln Arg Ser Arg ArgMet 450 455 460 Ile Val Val Leu Ser Pro Asp Tyr Val Thr Glu Lys Ser IleSer Met 465 470 475 480 Leu Glu Phe Lys Leu Gly Val Met Cys Gln Asn SerIle Ala Thr Lys 485 490 495 Leu Ile Val Val Glu Tyr Arg Pro Leu Glu GlnPro His Pro Gly Ile 500 505 510 Met Gln Leu Lys Glu Ser Val Ser Phe ValSer Trp Lys Gly Glu Lys 515 520 525 Ser Lys His Ser Gly Ser Lys Phe TrpLys Ala Leu Arg Leu Ala Leu 530 535 540 Pro Leu Arg Ser Leu Ser Ala SerSer Gly Trp Asn Glu Ser Cys Ser 545 550 555 560 Ser Gln Ser Asp Ile SerLeu Asp His Val Gln Arg Arg Ser Arg Leu 565 570 575 Lys Glu Pro Pro GluLeu Arg Ser Ser Glu Arg Val Ser Gly Ala Glu 580 585 590 Pro Ala Pro GlyThr Met Ser Lys His Arg Gly Lys Pro Ser Ala Ala 595 600 605 Cys Arg CysCys Val Thr Tyr Cys Glu Gly Glu Ser His Leu Arg Ser 610 615 620 Lys SerArg Ala Glu Met His Thr His Pro Gln Trp Glu Thr His Leu 625 630 635 640Cys Lys Pro Pro Leu Gln Glu Ser Glu Ser Gln Trp Ile Gln Asn Gly 645 650655 Thr Arg Pro Glu Pro Ala Pro Gln Ile Ser Ala Leu Ala Leu Arg His 660665 670 Phe Thr Asp Leu Ser Asn Asn Asn Asp Phe Tyr Ile Leu 675 680 685

What is claimed is:
 1. An isolated polynucleotide comprising SEQ ID NO:1 wherein the nucleic acid at 1792 is A or C.
 2. An isolatedpolynucleotide comprising a nucleic acid that encodes a polypeptidecomprising SEQ ID NO: 2, wherein the amino acid 598 is Thr or Pro
 3. Anisolated polynucleotide comprising SEQ ID NO:
 3. 4. An isolatedpolynucleotide comprising a nucleic acid that encodes a polypeptidecomprising SEQ ID NO:
 4. 5. An isolated polynucleotide comprising amolecule selected from the group consisting of: a) A polynucleotide thatencodes a polypeptide comprising amino acid residues 384-687 of SEQ IDNO: 2, wherein the amino acid at 598 is Thr or Pro; b) A polynucleotidethat encodes a polypeptide comprising amino acid residues 379-687 of SEQID NO: 2, wherein the amino acid at 598 is Thr or Pro; c) Apolynucleotide that encodes a polypeptide comprising amino acid residues389-685 of SEQ ID NO: 4; d) A polynucleotide that encodes a polypeptidecomprising amino acid residues 379-685 of SEQ ID NO: 4; e) Apolynucleotide that encodes a polypeptide comprising amino acid residues449-687 of SEQ ID NO: 2, wherein the amino acid at 598 is Pro or Thr; f)A polynucleotide that encodes a polypeptide comprising amino acidresidues 449-685 of SEQ ID NO: 4 g) A polynucleotide that encodes afragment of a polypeptide described in (a-g), wherein the fragmentinteracts with a signal transduction factor; h) An isolated nucleic acidmolecule that hybridizes to either strand of a denatured,double-stranded DNA comprising the polynucleotide of any one of a)-g)under conditions of moderate stringency in 50% formamide and 6×SSC, at42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SDS; i) Anisolated nucleic acid molecule that encodes a polypeptide that is atleast 85% identical to the polypeptides described in a)-g), wherein thepolypeptide interacts with a signal transduction factor; j) Apolynucleotide that is degenerate to any of the polynucleotides ofa)-i).
 6. An expression vector comprising a polynucleotide of claim 5.7. An expression vector comprising a polynucleotide that encodes apolypeptide comprising SEQ ID NO: 2, wherein the amino acid residue at598 is Pro or Thr.
 8. An expression vector comprising a polynucleotidethat encodes a polypeptide comprising SEQ ID NO:
 4. 9. A host cellcomprising the vector of claim
 5. 10. A process of preparing apolypeptide, the process comprising culturing a host cell of claim 9under conditions promoting expression of the polypeptide.
 11. A processof preparing a polypeptide, the process comprising culturing a host celltransformed with a vector of claim 7 under conditions promotingexpression of the polypeptide.
 12. A polypeptide selected from the groupconsisting of: a) A polypeptide comprising SEQ ID NO: 2, wherein theamino acid at 598 is Thr or Pro; b) A polypeptide comprising SEQ ID NO:4. c) A polypeptide comprising amino acids 449-685 of SEQ ID NO: 4; d) Apolypeptide comprising amino acids 449-687 of SEQ ID NO: 2, wherein theamino acid at 598 is Thr or Pro; e) A polypeptide comprising amino acids384-687 of SEQ ID NO: 2, wherein the amino acid at 598 is Thr or Pro; f)A polypeptide comprising amino acids 379-687 of SEQ ID NO: 2, whereinthe amino acid at 598 is Thr or Pro g) A polypeptide comprising aminoacids 379-685 of SEQ ID NO: 4; h) A polypeptide comprising amino acids389-685 of SEQ ID NO: 4; i) A polypeptide comprising a fragment of apolypeptide of a)-h) wherein the fragment interacts with a signaltransduction factor. j) A polypeptide that is at least 85% identical toa polypeptide of a)-g), wherein the polypeptide interacts with a signaltransduction factor.
 13. An antibody that is specific to a polypeptideof claim
 10. 14. A method for screening for an agonist or antagonist ofIL-1 comprising: Contacting a polypeptide of claim 8 with an IL-1 familymember and an IL-1 receptor family member in the presence of a candidatecompound, and comparing the interaction of the polypeptide in thepresence of the candidate compound with the interaction in the absenceof the compound, whereby a compound the modulates the interaction of thepolypeptide is identified as an agonist or antagonist of the polypeptideof claim 8.